The Influence Of Transplantation Bone Marrow Mesenchymal Stem Cells Via Artery In Nephropathy Rat

Objective (s):1、To establish the model of nephropathy inducted by the adriamycin (ADR).As the same time Bone Marrow Mesenchymal Stem Cells (BMSCs) were isolated, predicated, cultured and fluorescent marked.2、Observe curative effect by inject marked BMSCs into caudal veinand renal artery paths. Also, explore the feasibility of transplantation via renal atery.Methods:Excise 40 male rats’left renal and inject adriamycin 2.5mg/kg into the caudal vein weekly in two weeks to establish the model of nephropathy. Then take tissue of kindey to have pathological examination. At the same time, extract BMSCs from femurs and tibias by two Sprague-Dawley(SD)rats. After separation and purification, take flow cytometry identification and mark the 4th generation BMCs with the adenovirus with green fluorescence. Take 36 rats which models were successfully established were randomly divided into three groups,12 rats in each group:Group ADR, Group M-A and Group M-V. Also,12 normal rats will be blank control group(Group N).In group M-A, in direction of observation by DSA, PE-10 tubes will be intubated to rat’s renal artery with general anesthesia. Then slowly inject 2 ml,1×106 pc/mL BMSCs suspension. In ADR group, inject 2ml normal saline(NS) in the same method as group M-A. In group M-V, inject 2 ml,1×106pc/mL BMSCs suspension from caudal vein. In group N, inject 2ml NS from caudal vein.Test the 24 hour urine protein,hemoglobin, blood urea nitrogen(BUN), serum creatinine(Scr) and serum alb-umin level at the 1st day,7th day and 14th day after the transplantation. Also, execute 4 rats from each group and take the tissue of kindey to have pathological examination. Observe the BMSCs distribution in kidney by FCFM, and test the expression situation of aquaporin 1 (AQP1) and aquaporin 2(AQP2).Results:Model established rat’s 24 hours urine protein have increased significantly and BUN,Scr have increased sharply.Hypoalbuminemia and anemia were obvious and showed, inflammation, glomerular focal segmental sclerosis, atrophy of renal tubular, the abscission of the epithelial of renal tubular in pathology and model establishing succeed. The BMSCs showed typical spiral growth feature. We get high purity BMSCs after subcultured. The cells were in long spindle shape, similar form and grow in spiral shape with microscope observation. Being tested by flow cytometry, the cell surface markers antigens of BMSCs.such as CD29,CD44 and CD90 were in high expression, in contrast CD11b,CD34 were in low expression. BMSCs had transfected by adenovirus and generate steady glow,75% green fluorescence expression rate sign cells.After treatments, anemia severity of Group M-A and M-V had at each survey points of the 7th day and 14th day were decreased obviously than group ADR.24 hour urine protein amount, BUN and Scr were decreased. Serum albumin were increased. The differences had statistical significance. Each index were more obvious in contrast of group M-A and M-V at the 7th day, the differences had statistical significance as well. In group ADR, each index changes have no statistical significance renal artery angiography. That showed no radiographiccontrast nephropathy had happened. The Pathological examination showed, in group ADR, Glomerular mesangial cells and ground substances had varied level hyperplasia, glomerular showed focal segmental sclerosis, renal interstitial had flake fibrosis and soaked by large amount of inflammatory cells. In group M-V, Glomerular mesangial cells and ground substances had slight level hyperplasia, in renal interstitial,focal fibrosis and soaked by small amount of inflammatory cells can be seen. In group M-A, the glomerular capillary loop were expanded, renal tubular had no obvious shrink, renal interstitial fibrosis was not obvious, the inflammatory reaction was slight.The FCFM showed:Group N and group ADR had no obvious green fluorescence at each time point. Group M-V and group M-A were observed increasing intension green fluorescence at the first day after transplantation, the fluorescence was most obviously at the 14th day. In group M-V, green fluorescence was only distributed in renal tubular. However, in group M-A green fluorescence was mostly distributed in renal tubular, and a small amount can be seen in glomerular mesangial area.Conclusion(s):1.It is feasible to establish models of nephropathy rat through excise one side kidney and induct by the adriamycin. Model establishing time and adriamycin amount will be decreased. It is a safe, effective and repeatable way to establish model.2.Rat’s BMSCs are easy to get, separate, predicate, culture and identification.3. BMSCs transplantation has good curative effect to cure the model of adriamycin nephropathy rat. The urine protein can be reduced and the serum albumin be increased shortly. The reduction of BUN and Scr anemia severity will protect the kidney function. It is a feasible way to reach renal artery by intervention tube from left. BMSCs transplantation from renal artery can lead to better curative effects.