Protective Effects And Mechanism Of Mesenchymal Stem Cells On Adriamycin-induced Nephrosis In Rats

Objectives:1. To isolate and culture rat bone marrow mesenchymalstem cells and identify of its biological characteristics.2. To set up adriamycin-induced nephrosis model by injection ofdoxorubicin hydrochloride.3. To observe the effect of BMSCs transplantation into the model ofadriamycin-induced nephrosis by electron microscopy，detection ofbiochemical, RT-PCR, Real time PCR, Western blot and ELASAtechnology. At the same time to explore protective effects and possiblemechanism of mesenchymal stem cells (MSC) on adriamycin-inducednephrosis in rats.Methods:1. To isolate, culture and identify of rats BMSCs: To isolatethe mononuclear cell layer through rat lymphocyte separation medium，cultured BMSCs with the way of density gradient centrifugationcombined with adherent screening，and combine with these two methodsthe flow cytometry instrumentation face logo, osteogenic andadipogenic induction to identify its biological characteristics. 2. To set up the adriamycin-induced nephrosis model: model wasinduced by injecting doxorubicin hydrochloride by7.5mg/Kg weightinto caudal vein of rats once，to determine the model was successed ornot after14d through integrated the results: the general performance,24-hour proteinuria，creatinine and other biochemical indicators andcombined with the HE staining and electron microscopy morphologicalof the kidney tissue.3. Protective effects and possible mechanism of mesenchymal stem cells(MSC) on adriamycin-induced nephrosis in rats:(1) experimentalgroups: model group；prednisone group；MSC group；normal controlgroup.(2) Using electron microscopy to observe podocyte structure ofexperimental groups after intervention4w. And using the dynamics ofconventional biochemical detection methods to observe theexperimental groups:24-hour proteinuria, creatinine and otherbiochemical indicators.(3) Using ELASA to detect the monocytechemokine-1(MCP-1) expression in the serum of the different time ineach experimental groups.(4) Using RT-PCR,Real time PCR andwestern blot technique dynamic observation of podocyte hole membraneproteins and cytoskeleton-associated protein of nephrin, podocin，synaptopodin, p21, MCP-1in all experimental groups,and its expressionof mRNA levels and protein levels at different time.Results:1. By the methods of density gradient centrifugation combined with adherent screening to isolated and cultured rat BMSCs were easilyisolated, cultured cells with stromal cell surface antigen characteristics,does not have the surface antigens characteristic of hematopoietic stemcells, high purity, high activity and multi-directional differentiationpotential, full compliance with the biological characteristics of ratBMSCs.2. The adriamycin-induced nephrosis was induced by injectingdoxorubicin hydrochloride after14days, integrated all results todetermine this model. It was proved that it was successful. They reachto the experimental requirements.3.(1) After the intervention4w, MSC group, prednisone group comparedwith model group, the kidney tissues of foot process fusion and microvillivariability decreased significantly, MSC can protective effects on thepodocyte structural damage caused by doxorubicin.(2) After infusionof MSC,24h urinary protein, creatinine, blood urea nitrogen, cholesterol,triglycerides and albumin in MSC group compared with model group hada large degree of improvement, the renal function in rats have beenprotected, in particular24-hour proteinuria have reduced significantly.(3) After the intervention2w, the serum MCP-1expression in MSC groupand prednisone group compared with model group was significantlydecreased,through after the intervention of4w and8w, MSC control ofthe inflammatory capacity declined, but compared with the levels ofserum MCP-1expression in model group was still lower.(4) After the intervention4w, the expression of synaptopodin and p21mRNA in MSCgroup, prednisone group compared with model group was upregulated.(5) After the intervention4w, Western blot analysis showed that theprotein expression of synaptopodin and p21in MSC group, prednisonegroup compared with model group was upregulated.Conclusion：1. High purity and activity BMSCs was isolated from thebone marrow successfully by the methods of density gradientcentrifugation combined with adherent screening.2. The adriamycin-induced nephrosis model could be inducedsuccessfully by injecting doxorubicin hydrochloride by7.5mg/Kg weightinto caudal vein of rats once.3. According to the results of this experiment, protective effects andpossible mechanism of mesenchymal stem cells (MSC) onadriamycin-induced nephrosis in rats: MSC infusion inhibits the renaltissue of MCP-1production, thereby reducing the inflammation of thekidney tissu; through enhanced p21expression inhibits cell proliferation；at the same time through maintaining the foot cytoskeleton proteinssynaptopodin structural integrity, eventually decreasing proteinuria，protecting the glomerular damage caused by doxorubicin, delayingdisease progression.