雷帕霉素防治腹膜透析大鼠腹膜纤维化及其机理研究

发布时间：2018-08-27 15:35

【摘要】：研究背景：腹膜纤维化迄今仍然是一个世界性的难题,是导致尿毒症患者被迫中断长期腹膜透析的最主要因素。在非生理性腹膜透析液的长期刺激下,就会导致腹膜纤维化的发生,其主要机理是腹膜间皮细胞从上皮细胞表型向肌纤维母细胞的转分化,即上皮间质转化(EMT)过程的发生,由此启动细胞增殖和胶原等基质的积聚等,主要信号转导涉及到Smad通路、PI3-K/Akt/mTOR途径等。雷帕霉素(rapamycin),通过与FK结合蛋白(FK-binding protein, FKBP)结合,阻断哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)的表达,进而抑制细胞生长与增殖。在肾、肝、心等多个脏器领域的研究中雷帕霉素在抗增殖和抗纤维化方面已经显示了重要作用。目前雷帕霉素在抗腹膜纤维化领域的作用也受到越来越多的重视。目的：明确雷帕霉素对于腹膜透析模型大鼠腹膜纤维化的防治效果,阐明雷帕霉素对于腹膜透析模型大鼠腹膜纤维化的可能信号转导分子机制。实验方法：动物层面,取45只正常SD大鼠,随机分为5组：正常组,生理盐水组,高糖组,低浓度雷帕治疗组,高浓度雷帕治疗组,6周后处死,评估腹膜功能，，取腹壁组织,行HE及α-SMA、TGF-β的免疫组化染色,分析各组腹壁纤维化情况；细胞层面,取正常SD大鼠腹膜,原代培养腹膜间皮细胞,取第3-4代细胞,予以不同浓度高糖(30,60,120mM)干预,及不同浓度雷帕霉素(’10,100nM)干预,作细胞划痕实验,同时于处理48h后提取蛋白,分析高糖对α-SMA及E-cadherin的变化,以及雷帕霉素对腹膜细胞EMT的影响。结果：动物层面：高糖组大鼠的腹膜功能相较正常组及生理盐水组明显下降,低浓度雷帕及高浓度雷帕均显著改善腹膜功能(P0.05)；HE染色及α-SMA、TGF-β免疫组化显示,高糖组呈现腹膜纤维化状态,低浓度雷帕及高浓度雷帕均显著改善腹膜纤维化状态,且高浓度相较低浓度效果更明显。细胞层面：高糖处理后细胞呈现EMT改变,表现为：α-SMA的表达量相较正常组明显升高,E-cadherin的表达量明显下降,迁移率明显增加,且呈浓度相关性(均P0.05)。雷帕明显改善EMT表现,且呈浓度相关性(均P0.05)。结论：通过动物层面及细胞层面验证了雷帕霉素对高糖引起的腹膜纤维化有明显的防治作用,在有效浓度范围内,其防治效果与剂量呈一定的正相关。验证了EMT在腹膜纤维化过程中的作用。雷帕霉素抑制EMT的机制主要表现为：抑制腹膜间皮细胞迁移,减少TGF-β的分泌,抑制成纤维细胞的形成,抑制细胞外基质形成。
[Abstract]:Background: peritoneal fibrosis is still a worldwide problem and the most important factor leading to the forced interruption of long-term peritoneal dialysis in uremic patients. The long-term stimulation of non-physiological peritoneal dialysate will lead to peritoneal fibrosis. The main mechanism of peritoneal fibrosis is the transformation of peritoneal mesothelial cells from epithelial cells phenotype to myofibroblasts, that is, the process of epithelial mesenchymal transformation into (EMT). The main signal transduction involved in the Smad pathway, such as PI3-K / Akt / mTOR pathway. Rapamycin (rapamycin), inhibits cell growth and proliferation by blocking the expression of rapamycin target (mammalian target of rapamycin, mTOR) by binding to FK binding protein (FK-binding protein, FKBP). Rapamycin has been shown to play an important role in anti-proliferation and anti-fibrosis in kidney, liver and heart. At present, the role of rapamycin in the field of anti-peritoneal fibrosis has been paid more and more attention. Aim: to investigate the preventive and therapeutic effects of rapamycin on peritoneal fibrosis in peritoneal dialysis rats and to elucidate the possible signal transduction molecular mechanism of rapamycin on peritoneal fibrosis in peritoneal dialysis rats. Methods: at animal level, 45 normal SD rats were randomly divided into 5 groups: normal group, normal saline group, high glucose group, low concentration rapa treatment group, high concentration rapa treatment group and high concentration rapa treatment group after 6 weeks, the peritoneal function was evaluated and the abdominal wall tissue was taken. HE and 伪 -SMA-TGF- 尾 immunohistochemical staining were used to analyze the situation of abdominal wall fibrosis in each group. At the cell level, peritoneal mesothelial cells of normal SD rats were taken out, primary peritoneal mesothelial cells were cultured, 3-4 passage cells were taken and treated with different concentrations of high glucose (30 ~ 60120mm). And different concentrations of rapamycin (10100nM) were used for cell scratch test. After 48 h treatment, protein was extracted to analyze the changes of 伪 -SMA and E-cadherin and the effect of rapamycin on EMT of peritoneal cells. Results: at animal level, the peritoneal function of rats in high glucose group was significantly lower than that in normal group and saline group. The peritoneal function of rats in low concentration and high concentration of rapa improved significantly (P0.05) and 伪 -SMA-TGF- 尾 immunohistochemical staining. In high glucose group, peritoneal fibrosis was observed, and low and high concentrations of rapa significantly improved the state of peritoneal fibrosis, and the effect of high concentration phase was more obvious than that of low concentration. Cell level: after high glucose treatment cells showed EMT changes. The expression of 伪 -SMA was significantly increased compared with the normal group the expression of E-cadherin decreased significantly mobility increased significantly and showed a concentration correlation (P0.05). Rapa significantly improved EMT performance, and showed a concentration correlation (P0.05). Conclusion: rapamycin has obvious preventive and therapeutic effect on peritoneal fibrosis induced by high glucose by animal and cell level. In the range of effective concentration, the preventive and therapeutic effects of rapamycin are positively correlated with the dose. The role of EMT in the process of peritoneal fibrosis was verified. The mechanism of rapamycin inhibiting EMT is that it inhibits the migration of peritoneal mesothelial cells, reduces the secretion of TGF- 尾, inhibits the formation of fibroblasts and inhibits the formation of extracellular matrix.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.5;R656.41

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