【作者】 夏雨果；

【导师】 吴小候；

【作者基本信息】 重庆医科大学 ， 外科学， 2010， 硕士

【摘要】 第一部分目的建立大鼠移植肾纤维化加快模型并转染,观察shRNA-TGF-β1质粒(转化生长因子β1干扰质粒)对大鼠移植肾炎性细胞浸润及纤维化的影响。方法采用强化供肾冷缺血的方法,建立SD大鼠为供体,Wistar大鼠为受体的移植肾纤维化加快模型,将受体分为四组:T组为质粒组,受者注射RNAi质粒;H组为空质粒组,受者注射空质粒;Y组为移植组,受者仅行肾移植,不注射任何质粒;J组为假手术组,只打开腹腔切除左肾,不进行肾移植。并利用流体力学为基础的肾脏基因转染技术进行相应质粒的转染。HE染色评估各组移植肾炎性细胞浸润及纤维化程度。结果成功建立大鼠移植肾纤维化加快模型,移植组肾大鼠较短时间肾脏即纤维化缩小;HE染色示移植肾间质大量淋巴细胞浸润,细胞外充满大量的嗜伊红色细胞外基质,纤维化程度较重;相反质粒组的大鼠移植肾炎性细胞细胞浸润少,纤维化程度较轻。结论强化冷缺血能加快移植肾纤维化;以流体力学为基础的肾脏基因转染技术具有可行性;shRNA-TGF-β1质粒可减轻移植肾炎性细胞浸润及纤维化。第二部分目的:观察shRNA-TGF-β1质粒对大鼠移植肾TGF-β1及ECM(细胞外基质)表达的影响。方法:移植术后1、2、3个月时收集肾脏标本; RT-PCR及Western blot检测TGF-β1,Masson染色观察移植肾细胞外基质的沉积。结果:质粒组TGF-β1表达受到抑制,明显低于移植组及空质粒组(P<0.01 or P<0.05);质粒组移植肾细胞外基质沉积少于移植组及空质粒组。结论:shRNA-TGF-β1质粒能抑制移植肾TGF-β1的表达,减少细胞外基质的生成,一定程度上预防移植肾纤维化。第三部分目的:探讨shRNA-TGF-β1质粒对抗大鼠移植肾纤维化作用机制。方法:RT-PCR及Western blot检测各组移植肾p-Smad3(磷酸化-Smad3)、p-Smad7(磷酸化-Smad7)、E-cadherin、Ⅰ型胶原的mRNA及蛋白表达;E-cadherin及α肌动蛋白(α-SMA)免疫组化染色分别标记肾小管上皮细胞及成纤维母细胞,观察移植肾上皮细胞及成纤维母细胞变化的情况。结果:质粒组p-Smad3表达受到抑制,低于移植组及空质粒组(P﹤0.01 or P <0.05);质粒组p-Smad7表达上调,高于移植组及空质粒组(P﹤0.01 or P < 0.05);质粒组Ⅰ型胶原表达受到抑制,明显低于移植组及空质粒组(P<0.01 or P<0.05);质粒组E-cadherin表达高于H、Y组(P﹤0.01 or P < 0.05);质粒组E-cadherin标记的肾小管上皮细胞多于对照组,α-SMA标记的成纤维母细胞少于对照组,发生上皮-间质转分化的细胞较少。结论: shRNA-TGF-β1质粒对抗移植肾纤维化的机制可能是下调信号蛋白p-Smad3的表达、上调p-Smad7的表达,维持E-cadherin的表达,进而抑制了上皮-间质转分化,减少了包括Ⅰ型胶原在内的细胞外基质的生成。更多还原

【Abstract】 PART ONEObjective: To construct rat Transplant Kidney-sclerosis accelerated model and transfected with the plasmid shRNA- TGF-β1, and to observe the inflammatory cells infiltration and fibrosis extent of shRNA-TGF-β1 plasmid on renal allograft.Methods: Adopot the enhanced ischemia reperfusion injury method, and the SD to Wistar rat Transplant Kidney-sclerosis accelerated model was constructed. The receptors were divided into four groups: Group T (plasmid group) injected with shRNA-TGF-β1; Group H (vacant plasmid group) injected with vacant plasmid; Group Y (transplantation group) injected with no plasmid; Group J (sham-operated group) only removed the right kidney with no transplantation. Transfected with the plasmid based on the hydromechanics. The pathological changes and infiltrated inflammatory cells were assessed by HE staining. Results: Rat Transplant Kidney-sclerosis accelerated model was constructed successfully, and they presented fibrosis in a short time. There were large amount of inflammatory cells infiltration and severe deposition of ECM displayed HE staining. There were also less infiltrated chronic inflammatory cells and extracellular matrix deposition in the plasmid group.Conclusion: Rat Transplant Kidney-sclerosis accelerated model could be constructed via enhanced ischemia reperfusion injury. Renal gene transfection technology based on hydromechanics is feasible. The plasmid could inhibit the fibrosis of rat renal allograft.PART TWOObjective: To investigate the inhibitory effcts of shRNA-TGF-β1 plasmid on the expression of TGF-β1 and ECM in rat renal allograft.Methods: Transplanted renals were collected at the first, second and third months after transplantation. The gene transcriptional level of TGF-β1 by RT-PCR and the protein variation was examined by western blot, and the ECM deposition was assessed by Masson staining.Results: In group T, the expression of TGF-β1 was inhibited by the plasmid, and it was significantly lower than group H/Y (P﹤0.01 or P<0.05). The ECM depositon of Group T was much less than Group H/Y.Conclusion: The shRNA- TGF-β1 plasmid could inhibit the expression of TGF-β1 and reduce the synthesis of extracellular matrix, which could prevent fibrosis of renal allograft at some extent.PART THREEObjective: To explore the possible mechanism on the inhibition of rat renal allograft fibrosis by shRNA-TGF-β1 plasmid.Methods: RT-PCR and Western blot were used to detect the expression of phosphorylated Smad3/7 (p-Smad3/7), type I collagen and E-cadherin. Immunohistochemical stainings of E-cadherin andα-SMA were used to label tubular epithelial cells and fibroblast respectively in order to assess the cells’change.Results: In the plasmid group, the signal proteins of p-Smad3 was down-regulated and p-Smad7 was up-regulated(P﹤0.01 or P<0.05); Type I collagen was also inhibited and its expression was lower than Group H/Y (P﹤0.01 or P<0.05); the expression of E-cadherin was higher than Group H/Y(P<0.01 or P<0.05). Epithelial cells were much more and fibroblast was much less than that of control groups, and there was less occurrence of EMT.Conclusion: Our results suggest that shRNA- TGF-β1 plasmid could prevent the fibrosis of renal allograft. The mechanism may be associated with its effects of down-regulating p-Smad3, and up-regulating p-Smad7, maintaining the expression of E-cadherin, leading to the suppression of epithelial-myofibroblast transdiferentiation and extracellular matrix synthesis.更多还原