Effects And Mechanism Of Mesenchymal Stem Cells On Adriamycin-induced Podocyte Skeleton F-actin

PartⅠExtraction, Culture and Identification of rat BMSCsObject: the available bone marrow mesenchymal stem cells obtained by density gradientcentrifugationMethod:1. thigh bone and shinbone are separated from SD rat under sterile condition. theavailable bone marrow mesenchymal stem cells obtained by density gradientcentrifugation.BMSCs is observed by inverted microscope2. osteogenic and adipogenic differentiation of BMSCs by Osteogenic induction reagentsand adipogenic reagents. osteoblast is stained with VonKossa reagents，adipogenic cellis staied with oil red O reagents.3. bone marrow mesenchymal stem cell surface markers CD29, CD34, CD45, CD90aretest by flow cytometry.Result：BMSCs are spindle, polygonal fibroblast-like morphology, size uniformity andwhorled growth. BMSCs have differentiated osteoblast and adipogenic cell. The surfacemarkers CD29and CD90are positive.otherwise, the surface markers CD34and CD45arenegative. the purity of BMSCs is95%Conclusion:the available BMSCs can be extracted by density gradient centrifugationPart Ⅱ Extraction, Culture and Identification of podocyteObject: To obtain the available podocyte by graded sieving method.Method:1. glomerular is separated by80μm,150μm,200μm sieve.glomerular isobserved under an inverted microscope 2. the glomerular is digested by IV collagenase.the digested glomerular plant type Icollagen-coated culture flasks. glomerular and cultured cell morphology was observedunder an inverted microscope.3. neprin and WT-1epitope of cell are detected by immunofluorescence.Result：the glomerular can be obtained by graded sieving method.the podocytes growfrom the glomerular digested by IV collagenase,which are cobblestone-like cells.thenephrin epitope distribute cytoplasm and cytomembrane.otherwise,the WT-1epitopedistribute around cyteblast.Conclusion: the available podocyte can be extracted by graded sieving method.Part Ⅲ Effects and mechanism of mesenchymal stem cellson adriamycin-induced podocyte skeleton F-actinObject: To discuss Effects and mechanism of mesenchymal stem cells onadriamycin-induced podocyte skeleton F-actinMethod:1.the group of Effects of mesenchymal stem cells on adriamycin-inducedpodocyte skeleton F-actin as following:Normal group:podocyteModel group: podocyte+ADRMSC pretreatment group: MSC+podocyte（coculture24h）+ADRMSC treatment group: MSC+podocyte+ADRDEX pretreatment group: DEX+podocyte（coculture24h）+ADRDEX treatment group: DEX+podocyte+ADRpodocyte skeleton F-actin is test by Texas Red–X phalloidin at12h，24h，48h，72h，5d.2. the group of mechanism of mesenchymal stem cells on adriamycin-induced podocyteskeleton F-actin as following: Normal group:podocyteModel group: podocyte+ADRMSC control group: MSC+podocyte（coculture24h）MSC treatment group: MSC+podocyte（coculture24h）+ADRDEX control group: DEX+podocyte（coculture24h）DEX treatment group: DEX+podocyte（coculture24h）+ADRPP38is detected by western blotting after adding ADR0min，1min，3min，7min，15min，30min，1h，3h，6h，12h，24h. afterwards, PP38above group is test bywetern blotting at7min.Result：1.results of podocyte skeleton F-actin of the above groups are as following:(1) the podocytes in model group have begun apoptosising from24h.but podocyteskeleton F-actin don’t change. podocyte skeleton F-actin has changed from48h inmodel group.the podocyte has been death until5d.(2) podocyte skeleton F-actin in MSC pretreatment group and MSC treatment grouphasn’t changed compared with normal group,as well as DEX pretreatment group andDEX treatment group.(3) the therapeutic effect of pretreatment group and treatment group to podocyte skeletonF-actin is similar.2.the result of pp38in all groups are as following:(1)the express of pp38range after adding ADR at1min，3min，7min，15min，30min.otherwise, the express of pp38is most at7min.It decreased gradually at1h，3h，6h，12h，24h.(2) the express of pp38in MSC treatment group decreases compared with modelgroup.however,It increases compared with DEX treatment group and MSC control group.Conclusion:1.MSC can prevent podocyte skeleton F-actin from ADR.2. the therapeutic effect of pretreatment group and treatment group to podocyte skeletonF-actin is similar.3. the protect of MSC and DEX in podocyte skeleton F-actin may get through pp38. Part Ⅳ Trace of BMSCs in the ADR nephritis ratObject: to trace of MSC in the ADR nephritis rat through Small animals living imagerdevice and histopathologyMethod:1.the group of Trace of BMSCs in the ADR nephritis rat as following:Blank control group:inject NS to normal ratNegative control group: inject no-dyeing BMSCs to normal ratsMSC control group: inject dyeing BMSCs to normal ratsMSC model group: inject dyeing BMSCs to ADR nephritis rat2. proteinuria during24h is collected after injecting ADR for14d.the rats in all group areput in Small animals living imager device at10min，1h，2h，5h，8h，24h，48h，72h，1w，2w，3w，4w.the three random rats are sacrificed at24h，48h，72h，1w，2w，3w，4w.the kidneys of sacrificed rats are put in Small animals living imagerdevice.then,the kidney are made into frozen section. frozen section are observed underfluorescence microscope. frozen section are made into HE section. HE section areobserved under inverted microscope.Result：1.the result of Surface fluorescence in rats:Surface fluorescence of lung in the normal and MSC control group disappear at2h.Surface fluorescence of living in the normal group express most at10min,and disappearat3w.otherwise, Surface fluorescence of living in the MSC model group express most at8h,and disappear at3w.2.the Surface fluorescence of kidney in the normal group express most at24h,anddecrease gradually at other times. otherwise, Surface fluorescence of kidney in the MSCmodel group express most at48h,and decrease gradually at other times3.the dyeing BMSCs in the normal group can be found in renal frozen section at24h,and,however,disappear at3w.otherwise, the dyeing BMSCs in the MSC model groupcan be found at all times. Conclusion: the BMSCs can run to the kidney.and the chemotaxis of ADR nephritiskidney is stronger than normal kiney.