Abeta42 mice, IBM
Musculoskeletal Pathology
Genetically Augmenting A?42Levels in Skeletal Muscle Exacerbates Inclusion Body Myositis-Like Pathology and Motor Deficits in Transgenic Mice
Masashi Kitazawa,Kim N.Green,
Antonella Caccamo,and Frank https://www.wendangku.net/doc/187371275.html,Ferla
From the Department of Neurobiology and Behavior,Laboratory of Molecular Neuropathogenesis,University of California,Irvine, Irvine,California
The pathogenic basis of inclusion body myositis (IBM),the leading muscle degenerative disease afflict-ing the elderly,is unknown,although the histopatho-logical features are remarkably similar to those ob-served in Alzheimer’s disease.One leading hypothesis is that the buildup of amyloid-?(A?)peptide within selective skeletal muscle fibers contributes to the de-generative phenotype.A?is a small peptide derived via endoproteolysis of the amyloid precursor protein (APP).To determine the pathogenic effect of aug-menting A?42levels in skeletal muscle,we used a genetic approach to replace the endogenous wild-type presenilin-1(PS1)allele with the PS1
M146V
allele in MCK-APP mice.Although APP transgene expres-sion was unaltered,A?levels,particularly A?42, were elevated in skeletal muscle of the double trans-genic(MCK-APP/PS1)mice compared to the parental MCK-APP line.Elevated phospho-tau accumulation was found in the MCK-APP/PS1mice,and the greater activation of GSK-3?and cdk5were observed.Other IBM-like pathological features,such as inclusion bod-ies and inflammatory infiltrates,were more severe and prominent in the MCK-APP/PS1mice.Motor co-ordination and balance were more adversely affected and manifested at an earlier age in the MCK-APP/PS1 mice.The data presented here provide experimental evidence that A?42plays a proximal and critical role in the muscle degenerative process.(Am J Pathol2006, 168:1986–1997;DOI:10.2353/ajpath.2006.051232)
Inclusion body myositis(IBM),the most prevalent muscle disorder among the elderly,is characterized by proximal and distal skeletal muscle weakness.1–3The clinical fea-tures of this disorder are characterized by muscle weak-ness and atrophy,with selective involvement of both proximal and distal muscle groups,including the quad-riceps,iliopsoas,triceps,and biceps muscles.In spo-radic IBM,the majority of patients usually exhibit proximal weakness,and the quadriceps are more severely af-fected compared to other lower limb muscles.3In hered-itary IBM,however,affected muscles may exhibit a more restricted focus.For example,the quadriceps may be selectively spared in certain autosomal recessive cas-es.4,5Histopathologically,both sporadic and hereditary IBM are characterized by atrophic muscle fibers and fibers containing rimmed vacuoles and abnormal protein aggregates,particularly amyloid-?(A?),which is derived via endoproteolysis of the amyloid precursor protein (APP),and hyperphosphorylated tau.2,6–10
IBM and Alzheimer’s disease(AD)share many patho-histological features including the buildup of aggregated proteins such as A?and tau.In this regard,IBM can also be considered as a proteinopathy.As in AD,the role of the A?peptide is unresolved,although evidence sug-gests that it plays an early and critical role in the muscle degeneration.IBM remains the only known condition in which A?accumulates pathologically outside the central nervous system,except for age-related macular degen-eration.11This distinction implicates a critical role for A?in the pathogenesis of IBM.A noteworthy difference be-tween two degenerative processes is the location in which A?accumulates.In AD,A?has been traditionally viewed to exert its pathological effects extracellularly where it builds up in amyloid plaques,whereas it is only found intracellularly in IBM.12However,recent studies have demonstrated that various assembly states of A?found intracellularly contribute to pathophysiological changes in AD as well,including our own studies in transgenic mice in which intraneuronal A?appears to induce deficits in synaptic plasticity and trigger the onset of cognitive decline.13,14Moreover,soluble oligomeric Supported by the National Institutes of Health(grant AG20335).
Accepted for publication February28,2006.
Address reprint requests to Frank https://www.wendangku.net/doc/187371275.html,Ferla,Ph.D.,Department of Neurobiology and Behavior,1109Gillespie Neuroscience Facility,Univer-sity of California,Irvine,Irvine,CA92697-4545.E-mail:laferla@https://www.wendangku.net/doc/187371275.html,. American Journal of Pathology,Vol.168,No.6,June2006
Copyright?American Society for Investigative Pathology
DOI:10.2353/ajpath.2006.051232
1986
A?is now considered to be a potent neurotoxic compo-nent,found in the AD brain,and the levels of A?oli-gomers in brain,unlike fibrillar A?levels,correlates well with cognitive decline.15–18It is important to note that there is evidence that this species of A?occurs intra-neuronally.19,20Taken together,these data suggest a critical pathogenic role for intracellular A?in AD.
We previously developed a transgenic model of IBM by overexpressing the human Swedish APP mutation in skeletal muscle under the control of the muscle creatine kinase(MCK)promoter.21These mice generate A?,al-though the predominant isoform is the less amyloido-genic A?40peptide.Although A?42is considered more pathogenic in AD,it remains unclear whether it is more pathogenic in skeletal muscle compared to A?40.Here we used a genetic approach to selectively increase A?42 levels.Mutations in the presenilin-1(PS1)gene associ-ated with familial AD(FAD)are well known to modulate ?-secretase function to selectively increase the formation of the more amyloidogenic A?42peptide in neurons.22,23 Notably,this effect is not limited to neurons because FAD mutations in PS1are also known to significantly augment A?42levels in various cell culture models and transgenic animals.22,24,25We report that MCK-APP mice harboring the PS1
M146V
knock-in mutation(MCK-APP/PS1)produce markedly higher levels of A?42than the parental MCK-APP mice.The double transgenic mice develop his-topathological features resembling IBM,including centric nuclei,intracellular accumulation of A?peptide,and en-hanced inflammation around affected muscle fibers.No-tably,elevated A?42levels further lead to increased phosphorylation of tau in skeletal muscle.We also find that enzymatic activity for cyclin-dependent kinase5 (cdk5)and glycogen synthase kinase3?(GSK-3?)are elevated.These pathophysiological changes lead to an earlier onset of motor deficits in the MCK-APP/PS1mice. These results implicate an integral role for A?42in the progression of muscle degeneration,and suggest that A?-directed therapies may be effective for the treatment of human IBM patients.
Materials and Methods
Generation of Double Transgenic Mice Hemizygous MCK-APP mice(of the A6line)were
crossed to homozygous PS1
M146V
knock-in(PS1-KI)
mice to generate F
1
offspring.21,26PS1-KI mice,main-tained on a C57BL/6background like the MCK-APP transgenic mice,are homozygous for the mutant
PS1
M146V
allele.Because the mutation was knocked-in, expression of the mutant PS1protein is under the tran-scriptional control of the endogenous promoter,thereby ensuring expression in skeletal muscle.
For biochemical analyses,skeletal muscle tissues from nontransgenic(wild-type C57BL/6background:non-Tg), PS1-KI,single transgenic MCK-APP,and double trans-genic MCK-APP/PS1mice were homogenized in T-PER extraction buffer(Pierce,Rockford,IL)in the presence of protease inhibitor cocktail(Roche Applied Science,Indi-anapolis,IN)and phosphatase inhibitors(5mmol/L so-dium fluoride and50?mol/L sodium orthovanadate).The detergent-soluble fraction was isolated by centrifugation at100,000?g for1hour at4°C.The resultant pellet was homogenized in70%formic acid followed by centrifuga-tion at100,000?g for1hour at4°C to isolate the detergent-insoluble fraction.
Expression Analysis
Calf muscles,quadriceps,and triceps were dissected from4-month-old MCK-APP/PS1double transgenic mice and controls(PS1-KI),and total RNA was isolated using TRI reagent(Molecular Research Center,Cincinnati, OH).To determine levels of human APP expression,iso-lated RNA(10?g)was analyzed by Northern blot using 32P-labeled0.24-kb simian virus40(SV40)poly(A)DNA fragment as described previously.21Briefly,the human APP transgene was constructed with the use of the MCK promoter so that gene expression would be targeted to skeletal muscle.The transgene also included the polyad-enylation signal from SV40.Because the SV40sequence is not present in PS1-KI mice,this sequence can serve as a specific probe to detect the human APP transgene mRNA product selectively in the transgenic mice.Equal RNA loading was confirmed by probing for GAPDH (Ambion,Austin,TX).
Immunohistochemical Analysis
Skeletal muscle tissue was snap-frozen in liquid nitrogen-cooled isopentane and stored at?80°C.Cryosections were cut at10?m,placed onto silane-coated slides,and stored at?20°C.Hematoxylin and eosin staining was performed to determine the general morphology of the muscle.Serial sections were immunostained to deter-mine the localization of APP and A?fragments.Mouse anti-human A?antibody6E10(Signet,Dedham,MA)was used to stain both human APP and A?-containing frag-ments,P2-1antibody recognizes full-length human APP (gift from Dr.William Van Nostrand,State University of New York at Stony Brook),and anti-mouse CD8anti-body(Serotec,Raleigh,NC)was used to detect activated inflammatory T cells. Immunoprecipitation
Immunoprecipitation was performed before the kinase assay.One hundred?g of skeletal muscles from6-and 14-month-old PS1-KI or MCK-APP/PS1mice were immu-noprecipitated with protein A-agarose(Calbiochem,La Jolla,CA)for cdk5antibody or protein G-agarose(Roche Applied Science)for GSK-3?or AT8antibodies overnight at4°C.The resultant protein-antibody-agarose complex was washed three times with0.5?STEN(25mmol/L Tris, pH7.6,75mmol/L NaCl,1mmol/L ethylenediaminetet-raacetic acid,and0.1%Nonidet P-40).For immunoblot-ting,the complex was then resuspended in2?loading buffer and incubated for10minutes at70°C.
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Immunoblot Analysis
Equal amounts of protein from each fraction were re-solved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(4to12%Bis-Tris gel from Invitrogen, Carlsbad,CA).After transfer onto nitrocellulose,the blots were incubated with the respective antibodies:6E10(1: 1000);anti-C-terminal fragment of APP(1:5000;Cell Sig-naling Technology,Beverly,MA);anti-PS1(1:500,Cell Signaling Technology);AT8(phosphorylated tau at serine202and threonine205;1:2000;Innogenetics, Alpharetta,GA);anti-cdk5(1:1000,Calbiochem);anti-GSK-3?(1:1000;BD Transduction Laboratories,San Diego,CA);anti-GSK-3?(phosphorylated at serine9, 1:1000;Cell Signaling Technology),anti-p38MAPK(1: 1000,Cell Signaling Technology),anti-phospho-p38 MAPK(phosphorylated at threonine180and tyrosine 182,1:1000;Cell Signaling Technology),anti-JNK(1: 1000;Cell Signaling Technology),and anti-phospho-JNK (phosphorylated at threonine183and tyrosine185, 1:1000;Cell Signaling Technology)followed by horserad-ish peroxidase-conjugated secondary antibodies.Protein bands were detected by enhanced chemiluminescence plus(Amersham Biosciences,Piscataway,NJ).Mem-branes were reprobed with antibody against GAPDH(1: 5000;Santa Cruz Biotechnology,Santa Cruz,CA)to con-trol for protein loading.
Enzyme-Linked Immunosorbent Assay(ELISA) A?Measurement
Both detergent-soluble and-insoluble fractions were used to detect A?40and A?42by ELISA as described previously.14,21,27MaxiSorp immunoplates(Nalge Nunc, Rochester,NY)were coated with antibody against A?1-17(gift from Dr.William Van Nostrand)at a concen-tration of25?g/?l,and A?40and A?42were detected by specific horseradish peroxidase-conjugated antibody against A?35-40(MM32-13.1.1)or A?35-42(MM40-21.3.4),respectively.
GSK-3?and cdk5Kinase Assays
Kinase assays were performed as described previous-ly.27Briefly,after immunoprecipitation with GSK-3?or cdk5antibodies,samples were mixed with50?l of reac-tion mixture containing20mmol/L MOPS,pH7.2,5 mmol/L MgCl
2
,1mmol/L sodium orthovanadate,5 mmol/L NaF,100?mol/L ATP,2.5?Ci[?-32P]ATP,and 0.2mmol/L cdk5substrate(Calbiochem)or0.2mmol/L GSK-3?substrate(Calbiochem).The reaction transpired for1hour at37°C,then35?l of supernatant was placed on Immobilon-nitrocellulose membrane(Millipore,Bil-lerica,MA).The membranes were washed in0.3%phos-phoric acid and counted in a scintillation counter to de-termine the kinase activity.Isolation of mRNA and Quantification of Inflammation by Real-Time Polymerase Chain Reaction(RT-PCR)
Total RNA was isolated from quadriceps of non-Tg,PS1-
KI,MCK-APP,and MCK-APP/PS1mice(14and24 months old)using TRI reagent(Molecular Research Cen-ter),and cDNA was synthesized using iScript cDNA syn-thesis kit(Bio-Rad Laboratories,Hercules,CA)as de-scribed previously.27Equal amounts of cDNA(?1?g) were subject to RT-PCR reaction for mouse CD8mRNA by iQ SYBR Green supermix(Bio-Rad Laboratories)us-ing primer pair of5?-TGT GAA GCC AGA GGA CAG TG-3?and5?-CAG GAT GCA GAC TAC CAG CA-3?. Cycle threshold(Ct)values were calculated by MyiQ software(Bio-Rad Laboratories),and the relative fold changes in mRNA were determined as relative to GAPDH mRNA levels in each treatment group(mouse GAPDH primer pair:5?-AAC TTT GGC ATT GTG GAA GG-3?and 5?-ACA CAT TGG GGG TAG GAA CA-3?).
Rotarod Motor Test
Motor performance was evaluated using the accelerating rotarod(Accuscan Instruments,Columbus,OH)as de-scribed previously.21Mice were placed on a rotating dowel and required to continuously walk forward to avoid falling off.The rod was accelerated throughout20sec-onds to a constant speed of10rpm,and each trial was ended at60seconds.Non-Tg,PS1-KI,MCK-APP,and MCK-APP/PS1mice(ages1to23months)were given10 training trials per day for2consecutive days,and probe trials were completed on the third day.In the probe trial, each mouse was tested five times and time of fall-off was recorded and averaged.
Statistical Analysis
All data were analyzed using one-way analysis of vari-ance or unpaired t-test,and P?0.05or lower was considered to be significant.
Results
Introduction of PS1
M146V
Allele Does Not Alter Transgene Expression and Steady-State Levels To test the phenotypic consequences of augmenting A?42levels in skeletal muscle,we used a genetic ap-proach to introduce a mutant PS1allele into the MCK-APP single transgenic line by crossing them to homozy-gous PS1-KI mice.Double transgenic mice,referred to as MCK-APP/PS1mice consisted of the following genotype:
homozygous for the PS1
M146V
allele and hemizygous for the MCK-APP transgene.It was first necessary to deter-mine whether the expression profile of the MCK-APP transgene was altered in the MCK-APP/PS1mice.We analyzed total mRNA from various tissues by Northern blot and found that,as in the parental MCK-APP trans-
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genic line,21expression of the human APP transgene was exclusively directed to muscle tissue (data not shown).We next compared the expression levels of APP in vari-ous skeletal muscles of the double transgenic MCK-APP/PS1mice to the same muscles from the parental PS1-KI or MCK-APP mice.As expected,human APP mRNA is only apparent in the double transgenic mice and not in the PS1-KI mice (Figure 1A).Likewise,the levels of the transgene transcript are comparable between the MCK-APP and MCK-APP/PS1mice,suggesting that the intro-duction of the PS1M146V mutation did not alter the ex-pression pattern or levels of the human APP transgene (Figure 1B).
We next determined the effect of introducing the PS1M146V knockin mutation on human APP steady-state levels in skeletal muscle.Notably,human APP steady-state levels were comparable between both mouse groups,indicating that the introduction of the mutant PS1gene did not alter the steady-state levels of the human APP protein (Figure 1C).As expected,no signal was detected in muscle from non-Tg and PS1-KI mice after probing with the human-specific APP antibody (Figure 1C).The levels of PS1in skeletal muscle were also ex-amined and compared by Western blotting among the non-Tg,PS1-KI,MCK-APP,and MCK-APP/PS1mice and revealed that there was no difference in the steady-state levels among the four mouse groups,indicating that the PS1M146V mutant protein was maintained at physio-logical levels and not altered by the MCK-APP transgene (Figure 1C).
We next determined whether the steady-state levels of the human APP protein changed as a function of age.We compared three ages:6,14,and 24months of age.Interestingly,the steady-state levels of the holoprotein as well as its proteolytic fragment C99differed as the mice aged.The highest level of holoprotein was detected
at
Figure 1.Expression of the transgene and steady-state levels of APP and PS1in skeletal muscle is not altered between MCK-APP and MCK-APP/PS1transgenic mice.A:Northern blot analysis of human APP transgene mRNA from calf muscle (C),quadriceps (Q),and triceps (T),of PS1-KI and MCK-APP/PS1mice reveals the human APP transgene is only expressed in MCK-APP/PS1mice.Quadriceps has the highest transgene expression levels among other muscles.B:Densitometric comparison of the level of human APP transgene expression between the single transgenic MCK-APP mice and double transgenic MCK-APP/PS1mice.Graph represents densitometric analysis of human mRNA band.No overall difference in the expression level is apparent,indicating that PS1-KI allele does not alter expression of the MCK-APP transgene in the MCK-APP/PS1mice.C:Immunoblotting of transgene products from calf muscle (C)and quadriceps (Q)of nontransgenic (non-Tg),PS1-KI,MCK-APP,and MCK-APP/PS1mice shows that the steady-state levels of APP detected by 6E10antibody is present in MCK-APP and MCK-APP/PS1mice but not in non-Tg or PS1-KI.Membranes were reprobed for GAPDH as a loading control.Graphs represent the intensity of APP (left )and PS1(right )in the immunoblotting.D:Age-dependent change in APP steady-state levels in the MCK-APP/PS1mice.Quadriceps from 6-,14-,and 24-month-old MCK-APP/PS1mice shows highest APP levels at 14months,and lower levels at 24months.C99levels were highest at 6to 14months and also decreased with age.MCK levels showed a similar trend,indicating that the reduction in APP steady-state levels is attributable to the decreased activity of the MCK promoter.Membranes were reprobed for GAPDH to control for equal loading.E:The steady-state levels of C99between the MCK-APP and MCK-APP/PS1mice at 14months of age.No apparent difference was detected in the C99levels,indicating no alteration of BACE cleavage by introduction of PS1mutation.
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?14months of age,and was markedly lower in skeletal muscle from 24-month-old mice (Figure 1D).This pattern was consistent with our previous finding in the single MCK-APP line.21This age-related decrease in transgene levels appears to be attributable to an age-associated decline in the activity of the MCK promoter,as we find that the steady-state levels of the endogenous mouse MCK protein are also markedly decreased at 24months (Figure 1D).Compared to the age-matched parental MCK-APP mice,C99levels were slightly increased in the MCK-APP/PS1mice at 14months of age although the difference did not achieve statistical significance (Figure 1E).Therefore,because the pattern and levels of the human APP transgene were not modulated by the intro-duction of PS1M146V gene,and the levels of PS1protein were also maintained at physiological levels,any change in the onset of the phenotype must be due to modulating APP processing to favor A ?42formation.
Augmenting A ?42Exacerbates the IBM-Like Pathology in Skeletal Muscle of the MCK-APP/PS1Transgenic Mice
A key question to resolve is whether the processing of the APP protein in skeletal muscle is altered in response to the introduction of the PS1mutation and to determine which A ?species,A ?40or A ?42,predominates.In our previous study,both A ?40and A ?42were produced in skeletal muscle of the parental MCK-APP transgenic mice based on SELDI-MS analysis,although it was clear that A ?40levels were much higher than A ?42levels.28This observation confirms that both A ?40and A ?42are produced in skeletal muscle of the MCK-APP mice and that these levels are markedly higher than those in age-matched non-Tg mice.28Because the goal of this current study was to specifically augment A ?42levels,we next quantitatively determined the levels of both A ?species in muscles of the MCK-APP/PS1mice by ELISA using end-specific antibodies against A ?40and A ?42.Protein ex-tracts were prepared from the quadriceps and calf mus-cle of 14-month-old non-Tg,PS1-KI control,MCK-APP,and MCK-APP/PS1mice.No detectable A ?40or A ?42was found in muscle from non-Tg or PS1-KI mice (data
not shown),whereas relatively high levels of detergent-insoluble A ?40and A ?42were detected in muscle from the MCK-APP/PS1mice (Figure 2,A and B).Furthermore,the ratio of A ?42/A ?40was markedly elevated in the MCK-APP/PS1mice compared to the parental MCK-APP mice (Figure 2C).Therefore,as in neurons,the introduc-tion of the PS1mutation in muscle shifted the processing of APP to favor the generation of A ?42and also in-creased total A ?production.
We further examined the age-dependent processing of APP in the MCK-APP/PS1mice.Both calf muscle and quadriceps were harvested from MCK-APP/PS1mice at 3,6,14,and 24months of age,and detergent-soluble A ?40and A ?42were measured.The levels of A ?40and A ?42in calf muscles and quadriceps increased in an age-dependent manner up to 14months of age but were lower at 24months (data not shown).This change corre-sponded to the lower APP steady-state levels that were observed at 24months of age (Figure 1D).
Having demonstrated that the introduction of the mu-tant PS1allele into the MCK-APP mice augments A ?42levels,we next histopathologically evaluated the double MCK-APP/PS1transgenic mice using several criteria.First,we compared muscle sections from aged mice stained with the general stain hematoxylin and eosin and compared results to the parental PS1-KI and MCK-APP transgenic lines.No alterations in muscle cytoarchitec-ture were apparent in PS1-KI mice and the muscle ap-peared normal with peripherally localized nuclei and in-tact smooth muscle linings even in mice as old as 24months of age (Figure 3,A and B).In contrast,muscle (calf and quadriceps)from MCK-APP/PS1mice exhibited abundant centric nuclei (Figure 3,C and D).A similar histological feature was also observed in age-matched MCK-APP mice muscle (Figure 3I).Centric nuclei are a general marker of muscle pathology often observed in muscle disorders,although they can occasionally be found in normal mouse and human tissue at a low fre-quency of ?1to 3%;centric nuclei are also a feature of IBM myopathology.29In addition,histological analysis revealed that a significant proportion of the muscle fibers in the MCK-APP/PS1mice were smaller in size compared to control mice.These affected muscle cells were
sur-
Figure 2.Augmenting A ?42production in skeletal muscle of MCK-APP/PS1mice.A and B:Both detergent-soluble and -insoluble fractions from calf muscle and quadriceps of 14-to 15-month-old MCK-APP (open bars )or MCK-APP/PS1(filled bars )mice were used to measure total A ?40and A ?42by ELISA using specific antibodies against A ?35-40(MM32-13.1.1)and A ?35-42(MM40-21.3.4),respectively.Significant (P ?0.05or less)increases in A ?40and A ?42were detected in skeletal muscle from MCK-APP/PS1mice.*P ?0.05or **P ?0.01as compared between MCK-APP and MCK-APP/PS1mice.Number of mice used:n ?6(MCK-APP)and n ?7(MCK-APP/PS1).C:The A ?42/A ?40ratio significantly increases in MCK-APP/PS1mice as compared to MCK-APP mice at 14months of age.*P ?0.05.
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rounded by a relatively large number of hematoxylin-positive nuclei,most likely inflammatory cells.
Another major pathological hallmark of IBM is immune infiltration.Consequently,we analyzed and compared muscle tissue from MCK-APP/PS1mice to age-matched PS1-KI mice (Figure 3E).We observed extensive num-bers of CD8-immunopositive T cells surrounding or infil-trated into muscle fibers from the MCK-APP/PS1mice (Figure 3F).Thus,whereas the single MCK-APP line showed neutrophil rather than T-cell infiltration,21by aug-menting A ?42levels in skeletal muscle of the MCK-APP/PS1mice,we have derived a model that better mimics the inflammatory response of human IBM by inducing T-cell https://www.wendangku.net/doc/187371275.html,ing immunohistochemistry,we showed that human APP was readily apparent in skeletal muscle using anti-body P2-1antibody.In contrast,no staining was ob-served in muscles from the PS1-KI mice (Figure 3G).Both MCK-APP and MCK-APP/PS1mice showed high levels of P2-1-immunopositive muscle fibers (Figure 3,H and J),confirming the buildup of the transgene product in certain muscle fibers.The numbers of P2-1-immunopositive muscle fibers were comparable between the two trans-genic mice.As a rule,these P2-1-immunoreactive fibers were relatively smaller in size compared to other nonre-active muscle cells.
Inclusion bodies are the defining hallmark feature of IBM and are believed to play an important
pathogenic
Figure 3.IBM-like histopathological hallmarks are evident in skeletal muscle from the MCK-APP/PS1mice.H&E staining of calf muscle (A )and quadriceps (B )from 24-month-old PS1-KI or calf muscle (C )and quadriceps (D )of 24-month-old MCK-APP/PS1mice.E and F:CD8immunostaining of quadriceps from 24-month-old PS1-KI mice and MCK-APP/PS1mice.F:Skeletal muscle from MCK-APP/PS1mice exhibits increased numbers of CD8-positive activated T cells around muscle fibers.Inset is a higher magnification view of CD8-positive T cells.G:Full-length human APP immunostaining (P2-1)of quadriceps muscle from 24-month-old PS1-KI.H:P2-1immunostaining of quadriceps from 24-month-old MCK-APP/PS1,confirming the presence of APP in muscle fibers,and APP-immunopositive fibers tend to be smaller in size compared to nonstained fibers.I and J:Calf muscle from 24-month-old MCK-APP mice was stained with H&E (I )or P2-1antibody (J )for comparison with MCK-APP/PS1mice.The histopathological staining shows similar histopathological patterns with MCK-APP/PS1mice shown above.K:Intracellular A ?staining in quadriceps of MCK-APP/PS1mice,and at higher magnification (L ).M:The percentage of centric nuclei-containing muscle fibers in the parental MCK-APP and MCK-APP/PS1mice at 14or 24months of age.The graph represents mean ?SEM of seven animals per group.N:The number of A ?-containing inclusions in the parental MCK-APP and MCK-APP/PS1mice at 14or 24months of age.Muscle sections were immunostained with 6E10antibody,and intracellular A ?inclusions were counted in random field.The graph represents mean ?SEM of seven animals per group.O:RT-PCR for CD8mRNA levels in quadriceps of MCK-APP or MCK-APP/PS1mice.The expression levels were individually normalized by GAPDH mRNA levels.The graph represents relative CD8mRNA levels to age-matched non-Tg or PS1-KI mice.There was no significant difference between non-Tg and PS1-KI mice.
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role in muscle degeneration.ELISA data showed that A ?42was predominantly produced in the skeletal muscle of the MCK-APP/PS1mice (Figure 2,A–C).We immuno-stained muscle with A ?-specific antibodies and observed prominent accumulation of A ?intracellularly.Quadriceps from aged MCK-APP/PS1mice showed numerous intra-cellular inclusions that were immunoreactive with the A ?-specific antibodies (Figure 3,K and L),indicating that the MCK-APP/PS1mice mimic this important pathological feature of human IBM.
We next quantitatively measured pathological hall-marks of IBM including centric nuclei,A ?inclusion bod-ies,and inflammatory responses between the MCK-APP and MCK-APP/PS1mice at 14and 24months of age.
Numbers of centric nuclei were markedly higher in skel-etal muscle of MCK-APP/PS1at 14months compared to age-matched parental MCK-APP mice (P ?0.05;Figure 3M).By the age of 24months,the relative percentage of centric nuclei-containing cells was not different between the two mouse groups (Figure 3M).Similarly,the number of A ?-containing inclusion bodies was greater in the MCK-APP/PS1mice than the parental MCK-APP mice (Figure 3N).Furthermore,we noted that inflammation,as determined by CD8mRNA expression,in skeletal muscle occurred earlier in the MCK-APP/PS1mice compared to the parental line.CD8mRNA levels increased approxi-mately two times in 14-month-old MCK-APP/PS1mice compared to the MCK-APP mice (Figure 3O).Taken to-gether,these results show that augmenting A ?42exac-erbates the IBM-like muscle pathology in the double transgenic mouse model.
Enhanced GSK-3?and cdk5Activity in Skeletal Muscle of the MCK-APP/PS1Mice
Hyperphosphorylation of tau and subsequent accumula-tion of tau tangles in skeletal muscles are a major patho-logical hallmark of IBM.We examined whether tau phos-phorylation was affected by the exacerbation of
the
Figure 4.Tau phosphorylation is elevated in skeletal muscle of MCK-APP/PS1mice.The steady-state levels of phosphorylated tau were examined using antibody AT8.Tau-immunoreactive band from calf muscles of 6-or 14-month-old non-Tg,PS1-KI,MCK-APP,and MCK-APP/PS1mice are shown.Data are representative of four mice per group per age,and mem-branes were reprobed for GAPDH to control for equal loading (data not
shown).
Figure 5.Increased tau phosphorylation in skeletal muscle of MCK-APP/PS1mice is mediated by GSK-3?and cdk5.Steady-state levels of JNK,p38-MAPK,cdk5,GSK-3?,and their phosphorylated states were examined from 6-(A )and 14-month-old mice (B ).Specifically,we determined whether each kinase was phosphorylated at selective residues known to affect their activity:threonine 183and tyrosine 185for JNK,threonine 180and tyrosine 182for p38-MAPK,and serine 9for GSK-3?.The graphs represent ratio of the intensity of phosphorylated state relative to total level of corresponding kinase.*P ?0.05or **P ?0.01compared to non-Tg and PS1-KI,and ??P ?0.01compared to MCK-APP.
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amyloid pathology in skeletal muscle at6and14months of age.No shift in the AT8-positive tau(phosphorylated at serine202and threonine205;S202/T205)band was apparent in either non-Tg or PS1-KI mice at all ages tested,indicating that the PS1
M146V
mutation alone did not alter the tau phosphorylation pattern(Figure4).No-tably,we found that the levels of AT8-positive tau were increased in the MCK-APP/PS1mice compared to the MCK-APP mice(Figure4).The difference in phosphory-lated tau levels between the MCK-APP and the MCK-APP/PS1mice was even more apparent at14months of age,where approximately fourfold more phosphorylated (AT8-positive)tau was observed in the MCK-APP/PS1 mice(Figure4).
Several kinases including c-Jun N-terminal kinase (JNK),p38mitogen-activated protein kinase(p38-MAPK),cyclin-dependent kinase5(cdk5),and glycogen synthase kinase-3?(GSK-3?),have been extensively evaluated for their role in the phosphorylation of tau in AD.30–33We examined whether these putative kinases were also involved in the phosphorylation of tau in skel-etal muscle,and whether modulating A?42levels in the MCK-APP/PS1mice altered their activation state.The steady-state levels of total JNK,p38-MAPK,cdk5,and GSK-3?were not significantly altered among the trans-genic mice at the ages tested(Figure5,A and B).We first examined the activation/inactivation states of these ki-nases by measuring the phosphorylation profile of each kinase at specific amino acid residues.JNK and p38-MAPK are activated when phosphorylated at threonine 183and tyrosine185or threonine180and tyrosine182,
respectively,34,35whereas phosphorylation at serine9in GSK-3?is considered to cause inactivation of its kinase activity.36,37At6months of age,we found lower levels of phosphorylated GSK-3?in the MCK-APP/PS1mice(Fig-ure5A),and the reduction was significantly different from the other three groups(P?0.01).At14months of age,a marked reduction of phosphorylated GSK-3?was also observed only in the MCK-APP/PS1mice(Figure5B). Interestingly,at14months of age,the activation of p38-MAPK was also significantly increased as detected by the elevation of phosphorylated levels in both the MCK-APP and MCK-APP/PS1mice(Figure5B).Phosphory-lated JNK levels were slightly decreased only in MCK-APP mice,whereas the levels in MCK-APP/PS1mice were comparable to those in the non-Tg or PS1-KI mice (Figure5B).
To further assess the involvement of these kinases in tau phosphorylation in the skeletal muscle of the MCK-APP/PS1mice,we examined the association of these kinases with tau in skeletal muscle.We first immunopre-cipitated proteins using the phospho-tau-specific anti-body AT8and determined which kinases were physically associated with phosphorylated tau in skeletal muscle. Markedly higher levels of cdk5were detected in6-month-old MCK-APP/PS1mice,whereas higher levels of GSK-3?were found at14months(Figure6A).Although increased activation of p38-MAPK was initially observed at14months,p38-MAPK as well as JNK were not co-immunoprecipitated with phosphorylated tau(data not shown),indicating that both p38-MAPK and JNK may be less involved in tau phosphorylation in skeletal muscle. Moreover,immunoprecipitation with antibodies cdk5or GSK-3?followed by immunoblotting with a tau antibody further confirmed the involvement of cdk5and GSK-3?in tau phosphorylation in skeletal muscle of MCK-APP/PS1 mice(Figure6B).
To further verify the activation states of cdk5and GSK-3?,we measured the activity of these kinases. GSK-3?activity in skeletal muscle from the MCK-APP/ PS1mice was significantly higher at14months of age (P?0.05),whereas cdk5activity was increased in the MCK-APP/PS1mice at6months of age(Figure6C). Although it was not significant,the GSK-3?activity at6 months in the MCK-APP/PS1was higher than PS1-KI mice,and it partially correlates with the reduction of phospho-GSK-3?levels(Figures5A and6C).Taken together,our results suggest that both GSK-3?and cdk5are likely to be the primary kinases involved in the phosphorylation of tau in skeletal muscle of MCK-APP/ PS1mice,and that JNK and p38-MAPK are not likely to play a major role.
Motor Impairment Associates with the Levels of A?42in Skeletal Muscle
To assess the effect of the PS1mutation and the sub-sequent enhancement of A?42levels on another func-tion,we directly compared the performance of the double transgenic mice to controls using the
acceler-Figure6.GSK-3?and cdk5are activated and associated with phosphory-lated tau in skeletal muscle from the MCK-APP/PS1mice.A:Immunoblotting of cdk5and GSK-3?after immunoprecipitation with antibody AT8in skeletal muscle of6-and14-month-old PS1-KI or MCK-APP/PS1mice.Asterisk indicates higher kinase levels associated with phosphorylated tau in the skeletal muscle.B:Reverse immunoprecipitation/immunoblot further con-firms a physical association of cdk5or GSK-3?with phosphorylated tau (recognized by AT8).C:Kinase activity of cdk5(left)and GSK-3?(right) was measured from skeletal muscle of6-and14-month-old PS1-KI and MCK-APP/PS1mice.Data represent mean?SEM from four mice per group. *P?0.05compared to age-matched PS1-KI group.
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ating rotarod.The motor performance of non-Tg,PS1-KI,MCK-APP,and double transgenic MCK-APP/PS1mice was evaluated as a function of age.Five age groups were tested:1to 2,5to 6,11to 12,15to 18,and 21to 23months.Whereas the MCK-APP mice did not show impairments in motor performance until age 15to 18months,the MCK-APP/PS1mice showed sig-nificant (P ?0.05)impairment by 11to 12months of age (Figure 7A).Thus,augmenting A ?42levels in skel-etal muscle shifted the onset age of impairment by several months.The disparity in motor performance was most notable between the two groups at 15to 18months of age,as the MCK-APP/PS1mice were se-verely impaired compared to the single MCK-APP mice.By 21to 23months,the MCK-APP mice per-formed as poorly as the MCK-APP/PS1mice.Both non-Tg and PS1-KI did not exhibit any significant re-duction in motor performance at any of the ages tested.The impaired motor performance observed in the MCK-APP/PS1mice strongly correlated with the buildup of A ?42in skeletal muscle (r 2?0.6478;Figure 7B),whereas it was correlated in lesser degree with A ?40(r 2?0.2618;Figure 7C),providing corroborating evi-dence for a pathogenic role for A ?42in muscle degen-eration.In summary,we demonstrate that introduction of the mutant PS1M146V allele specifically increased the formation of A ?42and exacerbated the IBM-like phenotype.
Because there is a strong male predominance in IBM,we next evaluated male and female mice to determine whether there was a difference in performance between the sexes.Notably,we find that 11-to 12-month-old male
MCK-APP/PS1mice performed significantly (P ?0.05)poorer on the accelerating rotarod compared to age-matched female mice (Figure 7D).This disparity in motor performance was most apparent at this time point and diminished with age (Figure 7,E and F).Likewise,the male MCK-APP single transgenic mice were more se-verely impaired than the female counterparts at age 21to 23months (Figure 7F).Because the MCK-APP mice dis-play less pathological features of IBM than the MCK-APP/PS1mice,longer times are likely required for impairments to manifest in motor performance.In sum,we show that the phenotype of the MCK-APP and MCK-APP/PS1mice correlates with the level of A ?42in skeletal muscle,with the higher A ?42levels in the MCK-APP/PS1accelerating and exacerbating the phenotype relative to the MCK-APP mice.
Discussion
The accumulation of A ?within skeletal muscle fibers is one of the hallmark pathological features of IBM.Ab-normal accumulation of A ?-containing inclusions are present in skeletal muscle of IBM patients,and these inclusion bodies are found in nearly 100%of vacuo-lated muscle fibers.9,38Notably,these abnormal mus-cle fibers also accumulate presenilin-1,39which is con-sidered to be the catalytic subunit of ?-secretase,suggesting that dysregulation of APP processing is involved in the disease onset and/or progression.How-ever,it remains to be determined whether this buildup of A ?is an epiphenomenon/consequence of the
dis-
Figure 7.Augmenting A ?42exacerbates motor performance of the MCK-APP/PS1mice.A:Age-dependent reduction of motor performance in both the MCK-APP and MCK-APP/PS1mice.*P ?0.05or **P ?0.01as compared with non-Tg or PS1-KI.?P ?0.05or ??P ?0.01as compared between MCK-APP and MCK-APP/PS1mice.B and C:Correlation between accumulation of A ?42(B )or A ?40(C )in skeletal muscle and motor performance in MCK-APP/PS1mice.The x axis represents rotarod performance,and y axis represents A ?levels in skeletal muscle.D–F:Sex difference in motor performance was analyzed at age 11to 12months (D ),15to 18months (E ),and 21to 23months (F ).Open and filled bars represent males and females,respectively.For all experiments,numbers of mice were used in each group:n ?4to 7for 1to 2months,n ?4to 5for 5to 6months,n ?8(four males and four females)for 11to 12months,n ?8to 13(four to six males and four to seven females)for 15to 18months,and n ?8to 15(four to six males and four to nine females)for 21to 23months.*P ?0.05compared between male and female.
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ease process or whether it plays a more direct role in
contributing to the degenerative phenotype.Discrimi-nating between these two possibilities is not readily feasible by analyzing histopathological muscle sam-ples from affected human patients.Likewise,in vivo imaging methods have not yet reached the required point of sensitivity to address this question in living patients afflicted with the disease.
One means to better evaluate the role of A?in the pathogenesis of skeletal muscle disorders such as IBM is to use animal models.Because IBM is a chronic,age-related disorder like AD,we used an aggressive genetic approach to generate a mouse model that exhibits rele-vant pathology by overexpressing APP selectively in skel-etal muscle.Notably,there is evidence that APP mRNA levels are selectively enhanced in human IBM samples,40 thereby providing physiological justification for the over-expression of this protein in transgenic mice.We previ-ously generated a transgenic model of this myopathy by selectively targeting the precursor of A?(APP)to skeletal muscle fibers with the use of the MCK promoter.21These MCK-APP transgenic mice develop IBM-like pathology as well as an age-dependent motor impairment.The mice also predominantly produce the less amyloidogenic A?40isoform.However,it is well established,at least for brain amyloid disorders like AD,that the longer A?42 species is far more pathogenic.Consequently,here we used a genetic approach to modulate?-secretase activ-ity to favor production of A?42,by introducing a mutant PS1
M146V
allele into the MCK-APP transgenic mice.This goal was readily achieved by crossing the MCK-APP
mice to the PS1
M146V
knock-in mice.These double trans-genic MCK-APP/PS1mice produce significantly higher levels of the A?42peptide in skeletal muscle relative to single,parental line.Likewise,the A?42/40ratio is also significantly higher.Through analysis of the single and double transgenic lines developed here,we were able to determine whether A?40or A?42plays a more detrimen-tal role in skeletal muscle.As shown in the result,higher levels of A?42in skeletal muscle seem to exacerbate the phenotype,leading to the earlier manifestation of the IBM-like histopathological features and the motor impair-ment relative to the parental line.
Because the phenotype is exacerbated by the in-creased levels of A?42,it provides strong in vivo evi-dence that this peptide likely plays a pathogenic role in IBM,and likely not to be simply a marker or epiphenom-enon.It is critical to emphasize that the introduction of the mutant PS1allele did not enhance or alter the cellular profile of APP expression,thus the accelerated pheno-type is solely due to the modulation of APP processing and elevated levels of A?42.Higher A?42levels can have detrimental consequences for the AD brain,and our present findings demonstrate a similar finding is also true for skeletal muscle.Although our data show that aug-menting A?42levels in muscle accelerates the pathology and motor deficits,it does not address the mechanism by which high levels of the intracellular A?42peptide cause disease.
Several pathogenic mechanisms induced by misme-tabolism of APP have been proposed,and one or more of these mechanisms may underlie the exacerbated pheno-type we described here.Christensen and colleagues41 recently found a marked elevation of basal calcium stores in cultured myogenic cells overexpressing A?42,and they also found that the sensitivity of ryanodine receptors to caffeine was significantly increased in the presence of A?42in these cells.Although the exact consequences of altering calcium levels in skeletal muscle remain to be elucidated,their results indicate that calcium dysho-meostasis may be one of the pathogenic causes of IBM, and further work will be required to determine whether a similar alteration occurs in the transgenic mice.
Another potential mechanism that may be affected is the proteasome.APP/A?-mediated proteasome inhibition was recently reported by Fratta and colleagues.42They studied skeletal muscles from sporadic IBM patients and found that proteasome subunit(20S?)was co-localized with APP/A?,and its proteolytic activity was significantly reduced compared to healthy skeletal muscles.Further-more,cultured muscle fibers overexpressing APP showed a marked reduction of proteasome activity,and a proteasome inhibitor,epoxomicin,increased the forma-tion of inclusion bodies.42These data strongly suggest that pathological features of IBM including A?and hyper-phosphorylated tau-containing inclusion bodies and vac-uole formation may be partially due to proteasome inhi-bition caused by the abnormal buildup of A?in muscle fibers.
Interestingly,the MCK-APP/PS1mice showed a marked elevation of phosphorylated tau in skeletal mus-cle in an age-dependent manner.Increased levels of AT8-positive tau correlated well with production of intra-muscular A?levels and were likely mediated by the dif-ferential activation of cdk5and GSK-3?.Our current data strongly suggest that cellular mechanisms of tau pathol-ogy in this IBM model are similar to those in AD because both cdk5and GSK-3?play a critical role in the patho-genesis of neurofibrillary tangles.43–45In IBM-afflicted skeletal muscle,higher levels of cdk5have been ob-served,and it is co-localized with phosphorylated tau.46,47
Although the molecular basis that triggers the onset of IBM remains unknown,the findings of this study indicate that mechanisms underlying IBM may be similar to those in AD.In both disorders,A?seems to play a pivotal pathogenic role.Of course,the ultimate demonstration that A?is critically involved in the pathogenesis of IBM will await clinical trials.Given the remarkable pathobio-chemical similarities between AD and IBM,it will be in-teresting to determine whether A?-directed therapies that are in preclinical or clinical development for AD,such as BACE or?-secretase inhibitors or A?immunotherapy have therapeutically beneficial results for IBM patients as well.
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AJP June2006,Vol.168,No.6
工艺、技术管理制度
工艺、技术管理制度 1、目的 为规范生产工艺、生产技术的管理，进一步提高工艺、技术水平，结合实际，制订本制度。 2、适用范围 本制度通用于各生产、建设单位的工艺、技术管理。 3、准则 3.1 工艺、技术指标的管理 3.1.1 工艺指标由生产技术部制定报分管领导批准执行，任何人不得随意违反或改动，若因原料、设备的改变或因操作条件发生变化需作调整时，由有关车间以书面报告生产技术部审核、批准后执行。 3.1.2 继电保护整定值、仪表报警值的修订由车间提出申请，报生产技术部，召集有关专业技术人员讨论，并将结果上报申请批准执行。 3.2 工艺流程与设施的管理 3.2.1 对主要物料流程、辅助物料流程及其设施进行改变时，必须制定出完整的方案报生产技术部审核、批准后实施。 3.2.2 因扩建、技改及环保治理改变工艺流程及设施时，必须由有关单位写出可行性方案，经公司充分论证批准后方可设计施工。 3.3 工艺技术台帐管理 3.3.1 台帐种类包括：产量、消耗指标台帐，工艺指标台帐，技改、技措台帐，操作事故台帐，质量事故台帐。 3.3.2 台帐的具体内容 3.3.2.1 产量、消耗指标台帐要求：各车间都要建立产量、消耗指标台帐，对全部产品的日产量、各类消耗的日耗量、单耗等进行记录。 3.3.2.2 工艺指标台帐要求：各生产车间都要建立工艺指标台帐，对所有的厂控和主要非厂控工艺指标进行逐日记录。 3.3.3 技改、技措台帐 3.3.3.1 各单位均要建立技改、技措台帐。 3.3.3.2 内容包括技改名称、技改的内容（包括意义）、费用、实施的起止时间、提出人和实施人。 3.3.4 操作事故台帐 3.3. 4.1 各生产车间都要建立操作事故台帐。 3.3. 4.2 内容包括事故发生日期、地点、事故名称、事故属性、事故损失、事故原因、责任人、处理结果、防范措施，可根据本车间的实际情况增减内容。 3.3.5 质量事故台帐 3.3.5.1 各生产车间都要建立操作事故台帐。 3.3.5.2 内容包括事故发生日期、地点、事故名称、事故属性、事故损失、事故原因、责任人、处理结果、防范措施，可根据本车间的实际情况增减内容。 3.3.6 台帐的填写与保管 3.3.6.1 各车间工艺员必须按时认真填写，填写的数据必须准确、真实。 3.3.6.2 台帐必须清洁、无乱划。 3.3.6.3 每本台帐的封面都写清记录的起止时间，认真存档保管，不得损坏和
上海通用客户关系管理案例分析
上海通用客户关系管理分析 一、企业背景 上海通用汽车有限公司成立于1997 年，由上海汽车工业（集团）总公司、通用汽车公司各出资50% 组建而成。坚持“以客户为中心、以市场为导向”的经营理念，上海通用汽车不断以高质量、全系列的产品和高效优质的服务，以丰富、差异化的产品线满足日益增长的市场需求，成为“多品牌、全系列”汽车公司。上海通用汽车目前已拥有凯迪拉克、别克、雪佛兰，以及萨博四大品牌十八大系列近六十个品种的产品矩阵。各品牌在各自的细分市场中处于领先地位。 上海通用汽车公司认为如果能够在未来的环境之中生存下去的话，必须要有自己的核心竞争力，这个能力就是满足客户需求的能力，赢得客户的能力。上海通用始终从赢得客户能力的角度来进行战略性的调整。 通用公司通过在全球范围内实施CRM（客户关系管理）系统，有效地管理客户信息，并且赢得更多的客户，使得客户价值最大化。实施CRM系统是保证通用公司在50年后还能够生存的重要战略之一。 二、客户识别 通过长期的积累，上海通用公司有了非常丰富的客户信息资源。对于客户信息，通用公司一直是很重视的。在通用公司的内部，IT技术的应用同样是非常普遍的，在管理与生产的许多方面都应用了不同的IT系统。而且放在不同地方的客户数据能够共享，例如，销售人员的信息就能让维修服务人员来共享，不同品牌的客户信息资源也能够共享，通用拥有的几千个IT系统之间的沟通通过CRM 沟通比较流畅。 客户数据管理 集中管理客户信息。上海通用在过去也积累了很多的客户数据，例如客户购买汽车时的数据，包括客户是谁，地址、电话、邮政编码是什么，购买了什么型号
的汽车，车辆的发动机号码以及机架号码，以及从客户购车算起，至今这辆车的状况如何，汽车有没有进行过修理，如果是进行过修理的话，修了汽车的什么地方，更换了什么零部件，修理过程是在哪个维修站进行的，又是由谁来完成的等等汽车处于动态过程的数据。从而对车辆进行完整的了解，向客户提供更有针对性的服务。 潜在客户的开发。通用公司对潜在客户由着自己的定义，①从来没有买过车的人或者单位，现在打算买汽车，他们有可能购买通用的汽车；②没有买过通用公司汽车的人或者单位，通过做工作可以争取在他们购买新车时选择通用的产品。上海通用认为潜在客户开发的目标是要增加销售漏斗中潜在客户的流量，只有进入销售漏斗中的潜在客户数量增加了，从潜在客户转变为客户的数量才会增加。是以销售为动力的。不断的增加销售漏斗中潜在客户的流量，是一个循环往复的工作，不应该是阶段性的，或者时随意性的。 例子．．经过对以往数据统计分析，通用公司发现汽车展览会是吸引潜在客户的重要手段，有30%以上的客户是通过这种途径了解了通用汽车，并且成为购买通用汽车的客户，于是通用公司就在汽车展示过程中进行汽车的预定。在今年6月份举办的上海汽车展上，短短的3天时间里，订购赛欧汽车的人数就超过1,000人，订购新版别克GL汽车的有数百人。对潜在客户的研究发现，喜欢听歌剧的人对通用的汽车有兴趣，上海通用就在上海大剧院做促销活动，效果很好。 （2）潜在客户的管理。增加销售漏斗中的潜在客户流量，只是万里长征的第一步。将潜在客户成功的转化成客户，管理十分关键。上海通用将客户的购车时间分为：立刻购买、3个月内购买、6个月之内购买、1年之内购买这样几种类型，根据客户选择购买时间的不同，分门别类地采取不同的对应方法。例如对于一个立即购买的客户，系统就将这个信息送给销售人员，由销售人员进行及时的跟踪服务，对于3个月内购买的客户，系统会给销售人员提示，是不是可以将这个客户转化成立刻购买，提前客户的购买时间，对于6个月购买的客户提供比较详细的资料，对于1年之内购买的客户只提供普通的资料。通用的经验数据表明，选择考虑在3个月购买的潜在客户中，只有10％的客户会买车子，选择考虑在1年之内购买的潜在客户中，只有4％的客户会买车。 上海通用汽车公司销售流程图
IBM客户关系管理案例
IBM客户关系管理案例 一 Charles Schwab建立个性化服务 ●要点：在低费用的前提下提供个人化的服务 ●网站：https://www.wendangku.net/doc/187371275.html, ●位置：美国旧金山 ●贸易：股票贸易和投资 ● 1999年收入 (初步的)：39亿美元 ●上网始自： 1993年 ●注意点： 100家访问率最高的网站之一； J. D. Power 及其同事把 Schwab评为网上贸易领域中投资者满意度最高的网站。 作为贸易对消费者电子贸易的前驱者之一，Charles Schwab从1985年通过一个名为Equalizer的程序开始进行电子贸易。随后的几年中，Schwab增加了其他程序并在1996年推出了互联网化的服务项目--SchwabNow！由于这些服务都有其各自的规则和成本，这家公司就决定把这些服务结合起来并在1998年创建了https://www.wendangku.net/doc/187371275.html,。该公司制定了统一的贸易费用，并使其"砖瓦水泥"的公司完全支持它的网站并与其保持协调一致。 低费用，高贸易额 这一进入电子商务领域的积极进取的举措使Schwab能够获取最多的网上贸易额，并成为该领域早期的领导者。Schwab把贸易费用定为29.95美元(适用于达到1,000股的业务，接近于今天的标准)。这一费用大大低于以前的平均费用--80美元，但是这家公司知道，它能从更大的交易额中获得补偿。它确实实现了这个目的：Schwab现在控制了整个网上贸易的四分之一份额。 Merrill Lynch现在正迎头追赶Schwab，同时信息股本也增加了。以前，Schwab几乎不提供咨询服务。这一情况已发生了改变：今天，该网站的主页提供给消费者帮助、咨询和辅助信息。根据电子经纪业的副总裁Randy Goldman 所观察，这家公司正将重点从交易转移到客户关系上来。“我认为现在我们已经
XX公司技术质量管理体系(DOC 133页)
技术质量中心 部门名称： 技术质量中心 上级部门： 总经理 下级部门： 技术管理部、质量管理部、中心试验室 部门本职： 技术与质量管理 主要职能： 1．进行行业市场调查，收集产品开发与技术改进信息。 2．新产品的开发设计、现有产品的改进。 3．生产工艺文件的制定与更新、发放、登记、更换和归档管理。 4．新工艺、新技术、新材料的应用研究。 5．组织生产车间进行工艺技术、质量管理方面的知识、技能培训。 6．协助、指导生产车间解决技术问题、质量问题。 7．监督、检查和统计、上报生产车间的工艺纪律执行情况。 8．组织制订公司质量管理目标及各部门目标。 9．参与原辅材料供应厂商业绩的评审与考核。 10．产品质量检验，及检验结果的统计、分析与上报。 11．组织进行不合格品的评审，并监督处理结果。 12．检验、试验仪器设备的规划与管理。 13．质量事故的调查、分析和处理。 14．组织进行质量体系内部审核和质量改进活动，不断改进质量体系。
技术管理部 部门名称： 技术管理部 上级部门： 技术质量中心 下属岗位： 研发员、工艺员 部门本职： 产品开发与工艺、技术管理 主要职能： 1.制定产品开发及工艺技术管理有关规章制度并监督执行。 2.进行行业市场调查，收集产品开发与技术改进的信息。 3.新产品的开发设计。 4.现有产品的改进。 5.生产工艺文件的制定与更新。 6.工艺路线的优化、简化。 7.新工艺、新技术、新材料的应用研究。 8.组织实施生产车间的工艺技术培训。 9.协助、指导生产车间解决技术问题。 10.监督、检查和统计、上报生产车间工艺纪律的执行情况。 11.产品工时、材料消耗定额的制定与更新。 12.工艺技术文件的发放、登记、更换和归档管理。 13.本部门有关文件、记录的保管与归档管理。
中国移动集团客户关系管理系统解决方案
中国移动集团客户关系管理系统解决方案 集团客户是移动公司相对稳固的大客户群体和业务收入的要紧来源，具有丰富的、个性化和颇具规模的需求。集团客户服务是集团客户工作中最重要的一个方面。 集团客户往往不仅重视移动通信服务本身，还更看重服务的价值。依照客户需求，向客户提供深层次的整体服务解决方案，介入其内外部治理和运作，不但能够提高其自身的治理和运作效率，还加深了客户捆绑度；良好的集团客户服务，不但能够增加服务价值，还能够拓展我们的客户群。集团客户服务有利于提高我们深层次价值服务的水平和能力，制造与客户共赢的局面，从而有效幸免单纯的价格竞争，在目前严肃的价格战形势面前尤为重要。 做好集团客户服务工作，提高集团客户的忠诚度和中意度，并引导、激发其消费，能够有效地稳固和拓展高价值客户群，增加市场收入分额，提升企业核心竞争力，在猛烈的市场竞争中立于不败之地，需建设移动集团客户治理系统。 二、建设目标 1. 移动集团客户治理系统是移动业务支撑体系的一个有机组成部分，按集团公司集团客户两级系统、数据集中的原则进行建设。两级系统是指集团公司集团客户治理系统和省公司集团客户治理系统，数据集中是指集团客户相关数据要实现省级集中或部分数据的全国集中。移动集团客户治理系统的数据将全省集中。 2. 要支撑面向商业客户的售前、售中和售后的整个闭环生命周期。 3. 需要对全电信综合业务进行全面支撑。 4. 需要支撑集团客户的个性化资费和优待。 5. 移动集团客户治理系统必须表达企业的CRM理念，为集团客户提供整体解决方案，从而达到利用业务捆绑集团客户、留住集团客户、提高集团客户的中意度和忠诚度的目的。 6. 移动集团客户治理系统应智能地从数据中提取与集团客户相关的信息和知识，为集团客户人员制定客户服务、业务进展和市场竞争等策略，开展具体服务工作提供科学、准确、及时的指导。 7. 移动集团客户治理系统应满足各级业务单位集团客户工作治理要求，为集团客户经理及其治理人员提供有效的工作打算治理、任务治理、服务过程治理、职责权限治理和绩效治理手段，表达先进的现代企业治理思想，是企业核心业务流程在集团客户业务支撑方面的具体实现。 三、技术架构选择 依照移动业务的要求和和特点，移动集团客户治理系统应具备以下总体技术要求：
上海通用汽车crm
一、背景介绍 上海通用是上海汽车工业（集团）总公司和美国通用汽车公司各投资50%组建而成的迄今为止我国最大的中美合资企业，总投资为15.2亿美元。通用汽车已在国内建立了完善的生产、销售和服务体系，是目前国内最具竞争力的轿车生产和销售商。 汽车行业是竞争最为激烈的行业之一，越来越多的生产厂商意识到必须加快从生产型企业向服务型企业的转变，以客户的最新要求来指导生产，而不是要求客户的要求符合企业的产品。在汽车企业实施CRM应用中，上海通用汽车是一个成功的典范。在IBM公司的帮助下，2000年9月9日中国第一套企业级的CRM系统在上海通用正式运行。通用汽车选择全球最大CRM厂商Siebel的产品，实施方是IBM大中华区咨询与集成服务部。IBM全球服务与Siebel联盟已经为众多客户实施了Siebel系统，这也是上海通用选择IBM主要原因之一。 坚持“以客户为中心、以市场为导向”的经营理念，上海通用汽车不断以高质量、全系列的产品和高效优质的服务，以丰富、差异化的产品线满足日益增长的市场需求，成为“多品牌、全系列”汽车公司。上海通用汽车目前已拥有凯迪拉克、别克、雪佛兰，以及萨博四大品牌，形成凯迪拉克CTS、凯迪拉克SRX、凯迪拉克XLR；别克荣御轿车、别克君越轿车、别克君威轿车、别克GL8商务公务旅行车系列、别克凯越系列；雪佛兰景程轿车、雪佛兰LOVA乐风轿车、雪佛兰乐骋轿车、雪佛兰赛欧紧凑型轿车；萨博9-3运动型轿车、萨博 9-3高档敞蓬轿车、萨博9-5运动型高档轿车十八大系列近六十个品种的产品矩阵。在坚持品牌本土化和产品本土化的战略下，通过整合国际、国内资源，通过消化吸收式和整合资源的研发，上海通用汽车旗下各系列产品含有多项先进技术，在安全性、动力性、舒适性和环保方面表现优越，不仅在各自的细分市场中处于领先地位，并树立起新的标杆。 而这些成果的取得，离不开对客户关系的成功管理。 二、实施CRM前的概况 有人这样说：“第一辆车是依靠销售人员卖给客户的，而第二辆车以及以后的产品是依靠优质的售后服务卖出去的。” 在没有实施CRM以前，上海通用的销售状况如下： 其一、上海通用基本是按订单生产，但由于客户订单经常改变，往往造成供应商的被动。比如座椅真皮的颜色，为了适应客户的各种需求，供应商不得不每种颜色都准备一些。尽管如此，也避免不了出现某种颜色缺货或者积压的现象。如果使供应商及时看到客户需求的变化，从而及时调整他们的计划，就可能做到及时供货，也不会造成大量库存积压。因此，进一步的信息化已是势在必行。 其二、上海通用汽车通过不同手段和方式已积累了很多客户数据。但这些数据若从CRM角度来分析，会发现有些数据是残缺的，有些数据甚至是完全没有用的。例如在原系统中顾客购买汽车时的数据只包括姓名、地址、电话、邮政编码、所购汽车型号、发动机型号及机架型号，而顾客买完车后的车辆状况如何、有没有进行过修理、是怎么修的、在哪个维修站修的、都修了什么、更换了什么零部件、甚至具体到是哪位工人修的等一切数据都没有。一句话，有关已经卖出车辆的动态过程数据基本上没有，这对于上海通用汽车本部来说，就无法对车辆有一个完整的了解，更无法向顾客提供有针对性的服务。要知道，汽车是一种高价值的商品，对于厂商而言，了解汽车动态过程中的信息要比购买信息更为重要。 其三、顾客数据记录不科学。例如，上海通用汽车进行的电话营销活动有记录（为顾客生日寄贺卡，有表示关怀的记录），而顾客对产品或服务的投诉却没有记录。除此之外，有很多数据是分布在上海通用汽车内部各部门之间的，还有很多数据需要由分布在全国各地的零售商以及维修站来提供。 三、实施CRM流程 如下图1-1所示：
客户关系管理是什么
客户关系管理是什么 客户关系管理是企业用来管理客户关系的工具。客户关系管理是一个不断加强与顾客交流，不断了解顾客需求，并不断对产品及服 务进行改进和提高以满足顾客的需求的连续的过程。 客户关系管理的功能可以归纳为三个方面：市场营销中的客户关系管理、销售过程中的客户关系管理、客户服务过程中的客户关系 管理，以下简称为市场营销、销售、客户服务。 市场营销 客户关系管理系统在市场营销过程中，可有效帮助市场人员分析现有的目标客户群体，如主要客户群体集中在哪个行业、哪个职业、哪个年龄层次、哪个地域等等，从而帮助市场人员进行精确的市场 投放。客户关系管理也有效分析每一次市场活动的投入产出比，根 据与市场活动相关联的回款记录及举行市场活动的报销单据做计算，就可以统计出所有市场活动的效果报表。 相关销售 销售是客户关系管理系统中的主要组成部分，主要包括潜在客户、客户、联系人、业务机会、订单、回款单、报表统计图等模块。业 务员通过记录沟通内容、建立日程安排、查询预约提醒、快速浏览 客户数据有效缩短了工作时间，而大额业务提醒、销售漏斗分析、 业绩指标统计、业务阶段划分等功能又可以有效帮助管理人员提高 整个公司的成单率、缩短销售周期，从而实现最大效益的业务增长。 客户服务 客户服务主要是用于快速及时的获得问题客户的信息及客户历史问题记录等，这样可以有针对性并且高效的为客户解决问题，提高 客户满意度，提升企业形象。主要功能包括客户反馈、解决方案、 满意度调查等功能。应用客户反馈中的自动升级功能，可让管理者 第一时间得到超期未解决的客户请求，解决方案功能使全公司所有
员工都可以立刻提交给客户最为满意的答案，而满意度调查功能又可以使最高层的管理者随时获知本公司客户服务的真实水平。有些客户关系管理软件还会集成呼叫中心系统，这样可以缩短客户服务人员的响应时间，对提高客户服务水平也起到了很好的作用。 市面上很多的客户关系管理软件都会有很多其它功能，比如办公管理、行政管理、进销存等等，但是这些系统只是为使用者更加方便而产生的，其实与真正的客户关系管理没有任何的关系。 相关误区 案例 在选择客户关系管理软件时单纯以软件产品的案例作为软件采购的标准，甚至唯一标准。然而这对于IT技术日新月异变化的软件来说，这个标准往往让你选到的恰恰是过时的产品，甚至于即将被淘汰的产品。同样，在移动客户关系管理领域，技术的进步更是日新月异，智能机和3G的引入就是二零零几年出现的，显然基于非智能机和非3G环境的设计在技术的先进性上已经落后但是案例必然比前者多。此外，前几年的第一代的移动客户关系管理采用的是短信技术，第二代移动客户关系管理采用的WAP技术，这种技术已经被淘汰，它们的案例也一定比刚刚发展起来的采用WebService技术的第三代移动客户关系管理多的多，但是这种技术已经被淘汰了。在技术的进步以指数级增长的今天，一味地追求案例多，只会选择到一个技术上即将被淘汰的产品。因此选用软件第三条准则就是：绝对不要以案例多作为选择软件的唯一标准。 开发 自己开发客户关系管理更能够满足自身要求，还可以随时升级、维护，可控性强，能避免上当受骗，但实际上呢? 首先，客户关系管理系统已经涉及到越来越多的学科技术，包括计算机、通信、网络、管理与行为、多媒体、数据库、图形图像等等，是一个需要综合各种人才的团队工程，一个或者几个普通程序员已经很难做好
工艺管理制度范文
gz-scxb-15版本：a修改状态：0 XX年1月1日实施第一章总则第一条：为建立和健全工艺管理，加强生产责任制，严肃工艺纪律，以实现优质、高产、低耗、安全的目的，特制订本制度。第二条：工艺管理是生产技术管理的重要组成部份.其基本任务是：组织制定，执行各种产品的生产工艺技术规程及其它以此为中心的各项规程，督促、检查以生产工艺技术规程为中心的各项技术规程的执行情况，总结经验，改造生产技术，开展合理化建议的活动。并参与组织技术措施的实施；组织工艺查定和对工艺路线、工艺流程的技术经济评审，通过调查研究和生产实践，使各种规程制度不断得到修订和完善。第三条：工艺管理工作要同挖潜，革新、改造，以及新技术应用相结合，必须同提高产品质量，降低原材料消耗相结合；工艺管理还要与搞好环境保护，开展安全无事故活动相结合.第四条：工艺管理的日常工作：1、组织制订或修订产品或生产装置的工艺规程技术文件(并督促检查执行情况)。2、组织制订或修订各级工艺控制指标，操作指标，并督促检查执行情况。3、对现有的产品生产工艺，原材料消耗，以及中间控制进行定期的分析与评价，并提出改进意见和建议4、制订原始记录，工艺控制台帐、组织工艺技术分析5、开展合理化建议和技术改进活动。6、组织和参加工艺查定工作。7、配合技术情报，技术档案部门，收集和整理 国内外有关工艺技术资料。8、开展技术交流和协助开展技术教育活动。9、参加新产品技术鉴定工作。l0、参加制订和修订技术改造计划和长远规划工作。第二章工艺规程的编制和管理第五条：凡正式生产的产品和装置均需制订技术标准，工艺规程，岗位操作法和分析规程，均应该按规定的程序进行审查、批准，并给予受控编号后，方可贯彻执行.第六条：工艺规程是生产的法规，是各级生产指挥人员、生产技术管理人员开展工作的技术依据。工艺规程包括了产品的工艺路线和流程图，产品的质量，工艺和主要技经指标等基础资料。岗位操作法是按照工艺规程和生产实践经验组织编制的，是生产岗位操作人员必须共同遵守，执行的基本依据。工艺规程内容：一.产品概述1.产品名称、化学结构式、主要理化性质2.技术标准、产品质量规格、包装贮运方式3.主要用途、使用方法须知二.原辅材料1.原材料名称、规格及其主要指标检验方法2.辅助材料名称、规格及其主要指标检验方法3.其它材料名称、规格及其主要指标检验方法三.生产工艺过程1.工艺沿革(包括装置能力、技术进步等内容)2.化工工艺路线及其技术依据3.主要化学反应及副反应4.主要物料的平衡及流向5.工艺过程及流程图四.生产控制技术1.配方和配料(可列配方编号、配方另立)2.工艺控制点示意图3.各项工艺操作指标4.主要生产工序的控制方法5.中
盛天集团客户关系管理调研报告范本
盛天集团 客户关系管理调研报告 2015年4月9日
调研目的 1．认识什么是客户关系管理。 2. 认识客户关系管理的意义。 3. 认识客户关系管理的思路。 调研容 1．客观和全面介绍企业客户关系管理的做法。 2．分析和评价该企业客户关系管理做法的得与失。 3．为该企业的客户关系管理提出改进意见或思路。 调研意义 1.了解企业的客户关系管理。 2.分析企业的客户关系管理。 3.明确企业客户关系管理中的优缺点，并找出相关改进缺点的方法。
盛天集团发展概况 盛天集团来自，是一家以房地产开发、商业运营为主营业务，集钢材贸易、混凝土生产销售、物业管理服务等为一体的综合性企业集团。 盛天对诚信的坚守外化成企业对产品品质、服务品质的不渝追求，无论是地产还是其他经营活动中，始终以诚信为准则，狠抓质量，精益求精。最终演化出蕴涵独特太极精神的“诚信成就大业，品质决定品牌”企业口号， 一、企业客户关系管理发展状况 1.客户关系管理简介以及企业目标客户概况 客户关系管理是通过采用信息技术，使企业的市场营销、销售管理、客户服务和支持等经营流程信息化，实现客户资源有效利用的一套应用软件系统，其核心思想是“以客户为中心”，提高客户满意度，改善客户关系，从而提高企业的竞争力。 企业目标客户以年龄25-55岁为主，工作以在大中型公司、外资企业的白领阶层以及个体经济户为主，主要围绕自主购买和投资置业两个方面开展客户管理工作。 2.企业获取客户的做法 （1）体验式营销，推行不同户型不同风格的样板房，满足不同 消费者的需求，为顾客提供印象深刻的体验历程。 （2）发行期刊，对各期活动进行报道，以及对某个项目全方位
客户关系管理三要素
客户关系管理三要素 在大多数市场中，都有一两家公司因为同客户保持着更紧密的关系，而在业绩上远远胜出竞争对手。然而，这些企业的优势与客户关系管理（CRM）的工具和技术并无太大关系。事实上，IT技术仅仅是获得这一优势的一个必要但不充分的条件。 越来越多的证据表明，单靠IT本身，对于创造更好的客户关系并无多大助益。更大程度上，优异的客户关系能力取决于企业如何构建和管理它的组织，具体地说，它源自公司对三个组织要素的清晰聚焦和灵活安排。 第一个要素是组织定位，组织应将“留住客户”列为企业须优先考虑的事项，并且给予员工更大的自由度去满足客户的要求。 第二个要素是组织架构，包括组织的结构、为客户提供个性化产品和服务的流程，以及为督促员工致力于建立客户关系而采取的激励机制。 信息是最后一个要素，指的是深入的、相关性强的客户信息，而且是可以通过IT系统在全公司范围实现共享的。 所有公司都可以通过专注于这些关键要素，更清晰地感知它们之间的关联方式，从而改善自身的客户关系，并最终提高企业的经营业绩。要做到这一点，企业的管理者必须对每一个要素都要有更深的理解。 定位：“留住客户”优先 表明组织对客户关注程度的一个最重要的指标就是，全公司都拥有一个共同的信念：“留住客户”是企业每个人都优先关注的工作，而不仅是市场营销人员的事。另外一个指标是，有关客户的信息要在组织内开放共享。 如果某一职能部门（如销售部）认为它应独自拥有客户，那么企业的以上定位就达不到预期目的。有用的信息会被那些认识客户的人牢牢抓住，其他团队和部门都不大可能从他们那里分享到这些宝贵的客户资料。同样地，如果企业的思维定式和历史传统都鼓励员工依靠个人努力去赢取客户，那么就不会有人把更多的精力用在捕捉和集中共享客户信息上面。 以客户关系为中心的公司定位也会根据“区别对待不同客户”的理念而做出相应的调整。大部分公司都把这个提法挂在嘴上，但很少能做到像郭士纳（LouGertsner）领导下的IBM 那样，把服务于最好的客户并竭尽所能满足他们的需求列为公司的价值观。这种务实的方法使得IBM避免了遭遇惠普、思科和康柏都曾遇到的问题，它们都曾因追逐每一个互联网热点而忽略了自己的长期支付能力。通过在整个组织范围内强调客户保留的重要性，IBM脱颖
客户关系管理
复习题 一、填空题： 1．客户是把需求和利润带到我们面前的人，是企业生存和发展基础，客户的争夺是市场竞争的实质。 2.客户的状态有：潜在客户、目标客户、现实客户、流失客户。现实客户又分为：初次购买客户、重复购买客户和忠诚客户三类。 3.潜在客户是指对企业的产品或服务有需求和购买动机，有可能但还没有产生购买的人群。目标客户是企业经过挑选后确定的力图开发为现实客户的人群。 4.关系营销认为企业营销是一个与消费者、竞争者、供应商、分销商、政府机构和社会组织发生互动作用的过程，正确处理与这些个人和组织的关系是企业营销的核心，是企业成败的关键。 5.在交易的营销观念中，市场是由同质的无差别的个体客户构成，市场细分是在庞大的消费群中划分出同质性较高的目标受众；关系营销则认为市场中每个个体客户的需求和欲望、购买能力有着很大的差异，所以每个客户对于企业的价值也是不同的，不能将每个客户同等对待，应采取客户分级的方法来区别对待处于不同层念。 6.一对一营销不是一次关注一种需求，而是一次关注一位客户，尽可能多地满足这位客户的需求，关注的中心是客户。一对一营销不只关注市场占有率，还尽量增加每一位客户的购买额，也就是在一对一的基础上提升对每一位客户的占有程度，最终目标就是提升客户忠诚度，并使客户的终生价值达到最大化。 7.精准营销是依托信息技术手段，对客户的相关数据进行搜集，然后对这些数据运用技术平台进行统计和分析，掌握每一个客户的消费倾向，再通过电话、邮件等传播方式进行一对一的营销，并根据客户反映和市场效果进行不断地修改和完善。 8.客户关系管理是建立在信息技术和营销思想基础之上的一种先进的管理理念，是借助各种先进的技术手段和管理理念来研究与客户建立关系、提升关系和维护关系的科学，也是企业巩固及进一步发展与客户长期稳定关系的动态过程和策略，它将管理的视野从企业的内部延伸、扩展到企业的外部，是企业管理理论发展的新领域。 9.数据挖掘是从大型数据库中提取人们感兴趣的知识，这些知识是隐含的、未知的、潜在有用的信息，提取的知识表示为概念、规则、规律、模式等。 10．将最近一次消费与消费频率结合起来分析，可了解客户下一次交易的时间距离现在有多久；将消费频率与消费金额结合起来分析，可计算出客户为企业创造的利润。 二、简答题 1.客户信息的主要内容 个人客户信息的主要内容:基本信息;消费情况;事业情况;家庭情况;生活情况;教育情况;个性情况;人际情况。 企业客户信息的主要内容:基本信息;客户特征;业务状况;交易状况;负责人信息。 2.收集客户信息的渠道 收集客户的信息只能从点点滴滴做起，可通过直接渠道和间接渠道来完成。 直接收集客户信息的渠道，包括：在调查中获取客户信息，在营销活动中获取客户信息，在服务过程中获取客户信息，在终端收集客户信息，通过博览会、展销会、洽谈会等获取客户信息，网站和呼叫中心是收集客户信息的新渠道，从客户投诉中收集等。 间接收集客户信息的渠道，是指企业从公开的信息中或者通过购买获得客户信息，包括：各种媒介，工商行政管理部门及驻外机构，国内外金融机构及其分支机构，国内外咨询公司及市场研究公司，从已建立客户数据库的公司租用或购买等。 3.客户沟通的作用
工艺管理制度及考核办法
工艺管理制度及考核办法 1目的 为加强工艺管理工作，保证工艺技术的有效控制，特制定本管理制度及考核办法。 2适用范围 本规定适用于生产工艺技术管理控制。 3职责范围 生产技术部是工艺技术的归口管理部门，负责正常生产的工艺管理工作，组织工艺文件的编制，修订、发放及监督管理，负责工艺记录的审批登记管理，负责工艺文件执行情况的监督检查及考核工作。 4规定内容 4.1正常生产的工艺管理 4.1.1生产车间必须严格按生产工艺操作规程执行，并如实填写原始记录。 4.1.2为确保工艺指标的严格执行，生产车间主管要对工艺指标的执行情况进行检查，每天一次并做好记录，对查出的问题进行相应的处罚。 4.2 工艺记录的管理 4.2.1生产技术部是工艺记录的职能管理单位，负责对工艺记录的审核和备案工作。 4.2.2生产技术部对工艺记录要真实、及时、规范，并按月登记存放，保存一年，超过保存期的按程序要求进行及时销毁处理，作好记录。
4.3工艺文件的控制 4.3.1接收工艺文件的部门，必须按照《文件管理制度》要求，严格进行接收登记、借阅和发放，并有具体人员管理。 4.3.2工艺文件不允许复印和外借，车间主管要对工艺文件每月进行定期检查一次，制止文件的外流和丢失。 4.3.3车间主管将对发放的工艺文件进行检查监督管理，如发现文件管理不好，将对相关责任人进行相应处罚。 4.3.4工艺文件应保持完整，不允许随意涂改乱画及缺页现象。 4.3.5因工艺文件丢失或工作需要，要求补发工艺文件，应提交申请，写明原因，经生产技术部确认后，报总经理批准，方可发放。 5工艺管理考核办法 为了使工艺管理工作能够有效的运行，不流于形式，对于违反工艺管理规定的，将根据如下规定进行处罚。 a对于违反工艺管理规定，不按工艺安全操作规程严格进行操作，检查发现后给予批评，对第一责任人、当班班长进行罚款100元。 b因工艺文件管理不善造成丢失的，罚款100元；私自复印的发现一次罚款100元。 c对一月内造成指标不合格2次的操作工，月末加罚100元并下岗培训学习一周；对一月内造成指标不合格4次的操作工，月末加罚200元并调离本岗位。 d因不按规定操作而造成严重后果的，依据后果的严重程度、造成影响大小及发生的经济损失数额要素，对第一责任人作出下岗培训或开除等处罚。
以淘宝为例的客户关系管理中的客户问题【通用】.doc
以淘宝为例的客户关系管理中的“客户” 一、淘宝网客户关系特点论述 在淘宝网在线购物的运营模式下，其客户关系与传统模式相比呈现了新型的特征，主要体现在以下几个方面： 第一，淘宝网拥有比传统模式下更为庞大的客户群，其突破了空间的限制，源源不断的将买家吸引到这个巨大的平台上来。从单个卖家的角度来分析，除了拥有庞大的客户资源外，更可以依据淘宝平台所具有的数据管理技术区准确的分析市场和买家需求。 第二，从客户关系细分的角度来说，淘宝平台能够将买家进行过更加科学精确的细分，从而其有着精确地分类目录而满足不同客户的需求。 第三，淘宝网所建立的网络购物模式能够较传统模式带给客户更多的让渡价值。第四，淘宝网不仅包括了巨大的买家，也有着数目众多的卖家，他们有着不同的特色、信誉及品牌。单个的买家可挑选的空间大，流动性很强，单一的卖家较难建立客户的忠诚度和长期维持现有的客户。因此，卖家应该从特色、信誉、品牌、情感等多个角度来塑造自己的形象与定位。 第五，在淘宝中买卖双方可以进行实时的互动。淘宝通过平台技术实现了用户之间的即使沟通交流，可提升用户体验，同时也记录了用户信息的重要部分，提升用户满意度与忠诚度。 淘宝的运营平台，具备了实现良好的客户关系所必要的技术、资源、管理方法等基本前提，但同时也应该看到淘宝面临着激烈的竞争——如何让促进顾客忠诚？坚持以顾客为中心的理念，并不断的找到解决的措施才能够为淘宝提供持续的动力。
二．淘宝的客户关怀与保障 建立良好的企业形象，提高网络商品品牌知名度网络营销是建立在消费者的消
费动机的基础上，消费者的消费动机越强烈越容易产生消费行为。网络营销通过多维立体地宣扬企业及网络商品，扩大企业和企业产品的知名度，从而使网络营销获得成功。消费者如果在一些热门网站的广告中，频频见到某品牌的网络商品，势必增加其对该商品的注意和记忆，进而对这一网络商品产生良好的印象。消费时便会在情感上对这种商品有先入为主的感觉，极大地增加了消费行为发生的机率。 坚持虚拟商店的便利性，为消费者提供更好的服务向消费者提供快捷、便 利、周到的服务是每一个网络营销组织的宗旨，网络营销组织应增加通往购物消费网站的链接，保持消费者购物消费渠道的顺畅。同时应当保护消费者的利益，对商品质量予以保证，并与消费者在货款支付方式、送货及时与否、售后服务等方面，建立“一对一”的信息反馈联系。 三、淘宝客户满意度管理 1．淘宝的客户满意度评价 客户满意度哪怕只是略逊于“完全满意”，也会造成忠诚度大打折扣。如果大多数客户说自己对公司还算满意，但不是完全满意，管理者就应该警惕，要领会到其中的潜台词：客户对产品或服务的某些方面还是觉得不大称心，一旦有选择的余地，他们就容易受到诱惑，弃公司而去。而且无论客户感受如何，只要公司的产品或服务质量还不错，他们在满意度调查中就很难给出负面评价，结果造成客户在满意度调查中的评分通常偏高。事实上，我们只能认为打出5分（完全满意）的客户是非常忠诚的，而评分在3～4之间（满意）的客户很容易转向竞争对手。2．淘宝售后服务与保障 对于淘宝售后说明一般都要告知买家，首先是说明自己的产品的是否是正品、第二个就是产品的快递问题、最后就是产品的售后服务等内容。 您好！感谢您选购我们的商品和服务，谢谢您对我们的认可。 您的满意是我们的最终目标，您的鼓励是我们最大的动力，所以对我们好的一面请给予真诚的赞美吧，好东西需要大家来分享，还有更多的朋友需要您的引路导航，大家好才是真的好，所以请大方的亮出您对我们的评分吧！ 万一您对选购的商品不那么满意，请在付款之前及时拨打我店售后服务热线：0512-69370***。我们会认真听取您的意见并提供您满意的解决方案。如需换货，您仔细阅读以下内容：（1）淘宝用户名（请用正楷字）。（2）退换的商品名称及货号。（3）退换的原因： 如需退货,请注明所换宝贝的货号、颜色、数量如换新地址，请填写新地址（地址不变可不填） 注：1、退货寄回时间为收到货品的48小时以内，并填写好上面的表格将此卡片连同货品一并寄回我店。2.如果是商品质量问题，退换双程运费请您先垫付，我们将在收到商品后，通过支付宝或者银行转账支付给您。此外的费用需要您来支付。另由于路途遥远，在邮寄过程中外包装可能会产生一些小褶皱，没有影响到货品的，不在质量问题之列。 四．在客户上的现存问题 1现有客户资源利用率低，客户信息分散 目前，淘宝的客户信息还没有被作为企业重要的战略资源进行保护．网站与卖家
公司技术管理制度范本
技术管理制度 1总则 1.1技术管理是企业对生产全过程及其生产准备科学研究的全部技术活动进行科学管理的组织工 作，加强和完善技术管理，合理组织技术工作，建立严格的技术工作秩序，是现代企业生产的客观要求。 1.2技术管理是必须贯彻执行国家的有关法规政策，以经济效益为中心，节约能源，降低消耗， 提高产品性能和质量，提高企业职工技术素质。 1.3本公司实行总工程师领导下的各级工程技术人员的技术负责制，总工程师在公司总经理领导 下，负责全公司的技术管理工作，其他各技术管理部门在技术工作中受总工程师的领导。 2工艺技术 2.1工艺技术管理是技术管理的重要组成部分，其主要内容有工艺流程、工艺方法、工艺装备和 操作规程等。 2.2工艺技术管理的基本任务是认真编制、审核、生产工艺规程，并监督工艺规程在生产上的贯 彻执行。 2.3制定合理的工艺指标，保证安全生产、设备正常运行，产品质量合格，原材料动力消耗降低。 2.4工程部要深入生产生产一线，调查研究，协助车间解决工艺问题，根据需要召开技术分析活 动会，组织制定保证生产的技术措施；指导车间工艺技术管理工作。 2.5工程部要积极了解和吸收国内外同行业的先进技术，进行技术改造和科研活动，不断提高工 艺技术水平，鼓励职工进行技术革新，技术改造及开展合理化建议活动，对新工艺、新技术或改进性意见组织技术鉴定，提出措施并监督落实情况，推荐奖励项目。 2.6技术部协助总工程师做好技术的日常管理工作，制定工作计划，审核工艺规程，审核新产品， 新工艺、新技术在生产上的试行方案，负责工艺改革和变更的审批，检查执行情况等。 3工艺规程 3.1工艺规程是生产活动的基本技术文件，每个产品的生产都必须有批准生效的工艺规程作为依 据。 3.2工艺规程是组织生产的技术依据，必须严格遵守执行不得违反，检查贯彻执行工艺规程的部 门为工程部。 3.3工艺规程由，技术部编制、审核。 3.4经批准后的工艺规程有技术部负责发放登记，由使用部门专门保管，不得丢失，不得转让。 3.5工艺改进需要变更时，经技术部审核，经总工程师批准后方可变更。 3.6违反工艺规程进行操作或擅自变更工艺规程进行生产的要追究当事人的责任，由此引起安全 事故，损坏公司的资产或给公司造成不良影响的，要根据公司有关规定严肃处理。 5 技术培训 5.1 对于新招的职工或转岗工人上岗前，必须进行上岗前得技术培训。 5.2 技术培训的内容由技术部根据工作的具体要求确定，并纳入培训计划。 5.3 对参加培训的人员要进行考核，由工程部统一命题，办公室组织考试，考试合格者发放工作证。 5.4 技术部，根据公司的需要，可提出外出培训计划和人员，经总工程师批准后具体落实，拿到培训部门有效结业证件后到办公室办理存档手续，并进行登记。 6 技术情报 6.1 技术部负责各种科技图书、杂志及技术资料。产品设计等技术资料的管理、归档、登记工作。 6.2 需要借阅产品工艺技术文件。检验规程、产品标准以及产品设计、技术资料的人员，由总工签字批准后，方可借阅使用。 6.3 技术资料的复印、复制必须经总工签字批准后方可复印和复制。
广州白云机场集团客户关系管理方案
广州白云机场集团客户关系管理方案 第三章广州白云机场集团CRM现状分析 3.1广州白云机场集团概况 广州白云国际机场股份有限公司由广东省机场管理集团公司的优质资产组成，是经 营机场的专业公司。公司战略目标点明：要将白云机场建设成为依托泛珠三角地区，辐射东南亚和太平洋地区的综合性中枢机场，全面提升集团公司国际竞争力和盈利能力。自2004年转场运营以来，广州白云国际机场以每3年旅客吞吐量净增1000万人次 的增速发展：2004年即过2000万人次，2007年达到3000万人次，2010年突破4000 万人次，今年将排名世界15位左右。 白云机场已开通110多条航线，直达国内外100多个城市，保障机型近30种，与 国内外33家航空公司建立了业务关系。目前，中国南方航空集团公司和深圳航空公司都将白云机场作为公司的基地机场。 3.2广州白云机场目前的客户关系情况 广州白云国际机场股份有限公司是经营机场的专业公司，公司设11个职能部门，3 个管理部门，4个业务单位，9个经营公司，为国内外航空公司、旅客、货主提供服务，服务范围涵盖了航空地面保障、航空运输服务、航空护卫、物业管理、商业服务、餐饮业服务、广告业、设备维护服务等。如图3-1所示。 白云机场的直接服务对象是各家航空公司，此外，机场方面还有上百家的合作伙伴， 包括为航空公司提供直接服务的，如加油、机务维修、配餐等服务性公司，提供间接服务的场道维护公司、保洁公司。 3.2.1客户关系的定义 1．客户关系就是指客户与企业的关系，这种关系通过个人行为、心理、态度表现
出来。客户关系管理的目标是保持客户的长期满意度，促进企业的经济利益和长远发展。2．满意度：是指客户对服务或产品的感知到的结果和客户原有预期值之间的比 较，如果这种结果和预期相同，则可认为是满意，超过预期越多，满意度越高。它是对产品或者是服务性能，以及产品或者服务本身的评价，给出的与消费的满足感有关的快乐水平，是一种具有明显个人色彩和特征的评价。公司通过满足和超过客户的期望以及满足客户的需要来创造客户满意度。提高客户满意度不是企业的最终目标，企业必须在保证企业其他利益相关者，如股东、合作伙伴接受的基础上，努力为客户提高满意度。 3.2.2客户关系维护的原则 1．公司全体员工必须明确，公司的价值在于服务客户，公司为客户提供全方位的 服务，满足客户的需求。（见图3-5） 意识上：认认识到自己是在为客户工作，没有客户就失去价值； 行动上：应用反身原则，认清客户的真正需求，不仅使客户满意，而且要超出客户 的预期满意程度； 能力上：必须具备满足客户需求的相应能力； 2．信任与尊重客户，永远地视客户为朋友，给客户以可靠的关怀和贴心的帮助。 3．以客户需求为标准，根据客户要求提供保证质量的服务。 4．针对不同客户的需求制定与之相适应的物流方案。 5．建立顺畅有效的沟通。通过主动与客户接触，明确沟通目标，设计高效的营销 沟通，为客户提供充分的而信息和反馈通道。 6．对客户的查询，要在十分钟内给予明确的答复；对于投诉客户，要求在半个工 作日内妥善处理完毕。
客户关系管理
《客户关系管理》复习题 一、选择题 1、下列哪个抽样调查方法输入随机抽样（） A、任意抽样法 B、判断抽样法 C、等距抽样法 D、配额抽样法 2、一个完整的客户关系管理系统应不具有以下哪个特征（） A、开发性 B、综合性 C、集成性 D、智能性 3、对于企业来说，达到（）是基本任务，否则产品卖不出去，而获得（）是参与竞争取胜的保证。 A、客户忠诚，客户满意 B、客户价值，客户忠诚 C、客户满意，客户价值 D、客户满意，客户忠诚 4、（）是指与公司交易有较长的历史，对企业的产品和服务有较深的了解，但同时还与其他公司有交易往来的客户 A、新客户 B、常客户 C、忠诚客户 D、老客户 5、网络营销的关键在于把握（）这一核心问题，使营销网站真正成为连接企业外部信息（客户需求）与内部信息（客户信息的分析、决策）的接口 A、客户需求 B、客户满意 C、客户忠诚 D、客户价值 6、( )的意义体现在增加企业的盈利、降低企业的成本、提高企业的信誉度和美誉度等方面。 A、客户关怀 B、客户联盟 C、客户保持 D、客户忠诚 7、在客户识别中，下面哪种客户企业不必特别警惕（） A、只有一次购买历史的客户 B、过于自信、权力欲强的客户 C、对产品要求过高的客户 D、没有忍耐力的客户 8、CRM是（）。 A、销售自动化 B、客户信息管理 C、客户关系管理 D、客户关系营销 9、（）具体负责对CRM流程诊断与优化，并提交相应的CRM流程优化报告 A、项目经理 B、产品顾问 C、技术顾问 D、业务顾问 10、“货物售出，概不负责”，就是（）的典型说辞。 A、社会营销 B、市场营销 C、交易营销 D、关系营销 11、在日益激烈的市场竞争环境下，企业仅靠产品的质量已经难以留住客户，（）成为企业竞争制胜的另一张王牌 A、产品 B、服务 C、竞争 D、价格 12、著名经济学的2：8原理是指（）。 A、企业80%的销售额来自于20%的老顾客 B、企业有80%的新客户和20%的老客户 C、企业80%的员工为20%的老客户服务 D、企业的80%的利润来自于20%的老顾客 13、在客户满意度公式：C=b/a中，b代表的含义是（）。 A、客户满意度 B、客户对产品或服务所感知的实际体验