| Objective:1.To extract, culture and identify bone marrow mesenchymal stem cells(BMSCs) of Sprague-Dawley (SD) rats in vitro;and establish the model of adriamycin(ADR) nephropathy in rats for subsequent experiments.2.To investigate the effectiveness of BMSCs in curing adriamycin nephropathy rat, and explore the feasibility of transplantation via renal atery.Methods:1. BMSCs were extracted from the tibia or femur of the male SD rates, then identified and marked by the morphology of cells,the flow cytometry and the ossification and adipogenesis induction,et al.BMSCs were transfected by the adenovirus with green fluorescence,when there will be 80% of the 4th BMSCs fused, then made into the single cell suspension for use. The ADR model of nephropathy was induced by left nephrectomy plus injection of ADR(2.5 mg/kg) in SD rats via the tail vein.Then we should estimate whether the models were accomplished.2. To select 36 successful model rats with nephropathy and randomly divided into three groups:adriamycin nephropathic model control group(ADR,n=12),renal angiography and BMSCs transplanted group(M-A,n=12) and renal angiography and normal saline injected group(M-V,n=12).M-A group rats were transplanted BMSCs after angiography of the renal artery.N-A group rats were injected normal saline in the same way.ADR group rats are without any intervention.The serum creatinine,blood urea nitrogen,hemoglobin,serum albumin,serum sodium and 24h urine protein level were detected in the 1st day,the 7th day and the 14th day after the transplantation intervention.At that three time points,3-4 rats from every group were executed.Three or four rats were executed at the three time points.The renal morphology and expression of AQP1 and AQP2 were examined,and the distribution of labeled cells in the kidney was observed by confocal fluorescent microscopy.Results:1. The result of morphology of cells and identification:BMSCs have the typical characteristic of adherent growth,observed as the cells of spiral growth,uniform form, long shuttle type and high purity through the microscope after subcultured. CD90 and CD29 in the cell surface markers antigens of BMSCs were high expressed,however,the CD34,CDllb and CD45 are contrary.BMSCs are well differentiated by the inducement of ossification and adipogenesis.The expression rate of green fluorescence of BMSCs was high after them transfected by the adenovirus.The level of serum creatinine and blood urea nitrogen in this model are increasing dramatical ly,on the contrary,24h urine protein is decreasing which is the reason of hypoalbuminemia.The pathology of renal:glomerular focal segmental sclerosis, the atrophy of renal tubular, the expansion of tube cavity, the abscission of the epithelial of renal tubular,renal interstitial flake fibrosis and the inflammatory state are significant.2. One case was given up for the failure of intubation via left common carotid artery,one case died for excessive bleeding,22 cases were successfully operated,the success rate was 91.67%.The values of serum creatinine,blood urea nitrogen,24h urine protein and serum sodium were significantly lower in M-A group than those of ADR group in the 7th day and the 14th day(P< 0.05).So as the hemoglobin were significantly higher in the 7th day and the 14th day and the serum albumin were significantly higher just in the 14th day (P<0.05).However the values of serum creatinine,blood urea nitrogen,hemoglobin,serum albumin,24h urine protein serum and sodium level were not significantly different in M-A group than those of N-A group (P>0.05).The kidney organization structure stained by HE were observed under the optical microscope:The extra cellular matrix appeared different sizes of hyperplasia in the ADR group. There were glomerular focal segmental sclerosis, renal interstitial flake fibrosis and severe inflammation response.In the M-A group,the glomerular capillary loop had expansion. The renal tubules had no obvious atrophy. There were few inflammatory cells infiltrating and the renal interstitial fibrosis were slight. There were glomerular focal segmental sclerosis, atrophic renal tubules, renal interstitial flake fibrosis and a large number of inflammatory cells infiltration in the M-A group.Conclusion:1. The BMSCs of the SD rats can be acquired easily,the high purity stem cells that have the ability of multiple differentiations potential;the efficiency can be promoted by injecting Adriamycin and removing a single kidney to build the nephrectomy model;the BMSCs are qualified and can be used in the experiment.2. The technique of renal atery intubation via left common carotid artery is a feasible,effective way and can be successfully performed to complete renal angiography and stem cell transplantation.Transplantation of BMSCs can alleviate renal damage by inhibiting serum creatinine and blood urea nitrogen from increasing, balancing serum sodium,reducing the urinary protein excretion and improving the level of serum albumin. |
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