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目的:探讨骨桥蛋白(osteopontin,OPN)的shRNA干扰质粒在大鼠慢性移植肾肾病(chronic allograft nephropathy,CAN)模型中对大鼠移植肾上皮细胞向间充质转分化(epithelial to mesenchymal transition,EMT)的作用,初步探讨OPN在慢性移植物肾病中的作用及机制。方法:以F344大鼠为供体,Lewis大鼠为受体,行原位异体肾移植术,建立大鼠CAN模型(Allo组),以Lewis大鼠做供、受体作为同系移植对照(Syn组),以基于流体力学为基础的肾脏基因转染方法转染质粒到移植肾(RNAi组),采用空质粒作为对照(EV组),于术后12周处死各组动物,取移植肾标本进行HE和马松染色,观察组织形态学变化。采用免疫组织化学方法对移植肾组织α-平滑肌肌动蛋白(α-SMA)、E-钙黏蛋白(E-Cadherin)的表达进行观察。同时行Western blot检测移植肾组织OPN的表达,并对其灰度比作统计分析。结果:RNAi组OPN表达显著低于Allo组。Allo组和EV组大鼠移植肾组织形态学符合慢性移植肾肾病改变。RNAi组移植肾形态学变化较Allo组减轻。免疫组化结果显示,与Syn组相比,Allo组主要在肾小管上皮及肾间质强表达α-SMA,而肾小管上皮E-Cadherin表达较Syn组明显下降。与之相比,RNAi组α-SMA表达下调,E-Cadherin表达上调。结论:OPN在大鼠CAN模型移植肾中表达上调,OPN体内RNA干扰能够减少OPN在移植肾的表达,并且OPN的RNA干扰能缓解移植肾的病理学改变,抑制移植肾小管上皮细胞EMT。提示OPN与CAN的发生有潜在相关性,其可能通过促进肾小管上皮发生EMT途径影响CAN发病。
OBJECTIVE: To investigate the effect of shRNA interfering plasmid of osteopontin (OPN) on the rat mesenchymal transitional epithelial cells in rat model of chronic allograft nephropathy (CAN) EMT) to explore the role of OPN in chronic allograft nephropathy and its mechanism. METHODS: Lewis rats were used as recipients and Lewis rats were used as recipients. Allogeneic renal allografts were used to establish CAN model (Allo group). Lewis rats were used as donors. Syn Group). The plasmids were transfected into the transplanted kidney (RNAi group) by using the method of kidney gene transfection based on fluid mechanics. The empty plasmid was used as a control (EV group), and the animals in each group were sacrificed 12 weeks after the operation. HE staining and maston staining, histological changes were observed. The expression of α-smooth muscle actin (α-SMA) and E-cadherin in renal transplant recipients was observed by immunohistochemistry. At the same time, Western blot was used to detect the expression of OPN in renal allograft and the gray ratio was used for statistical analysis. Results: The expression of OPN in RNAi group was significantly lower than that in Allo group. The morphological changes of allograft kidneys in Allo and EV groups were found to be associated with chronic allograft nephropathy. The morphological changes of the kidneys in RNAi group were less than those in Allo group. Immunohistochemistry showed that compared with the Syn group, the Allo group mainly expressed α-SMA mainly in the renal tubular epithelium and the renal interstitium while the expression of E-Cadherin in the renal tubular epithelium was significantly decreased compared with the Syn group. In contrast, α-SMA expression was down-regulated and E-Cadherin expression was up-regulated in RNAi group. CONCLUSION: The expression of OPN is upregulated in renal transplant recipients with CAN. OPN RNA interference can reduce the expression of OPN in renal allograft. RNA interference of OPN can relieve the pathological changes of renal grafts and inhibit the EMT of transplanted renal tubular epithelial cells. It is suggested that there is a potential correlation between the occurrence of OPN and CAN, which may affect the pathogenesis of CAN by promoting the EMT pathway of renal tubular epithelial cells.