慢性肾脏

发布时间：2018-03-05 16:07

本文选题：内皮型一氧化氮合酶　切入点：4b/a　出处：《兰州大学》2016年博士论文　论文类型：学位论文

【摘要】：研究背景:各种慢性肾脏疾病不断进展的共同病理改变是肾小管间质纤维化,它是导致终末期肾衰竭的重要原因,也必然会给社会家庭带来沉重的“疾病负担”。这使得“慢性肾脏病-肾间质纤维化”成为近年肾脏病领域备受关注的焦点。慢性肾脏病肾间质纤维化与某些基因多态性相关,多个信号转导通路的活化介导着慢性肾脏病肾间质纤维化的发生发展。雷公藤甲素是雷公藤的主要活性成分,雷公藤虽然被广泛应用于多种肾脏病的治疗,但其作用机理有待于进一步探讨。目的:探讨内皮型一氧化氮合酶(eNOS)4b/a基因多态性与糖尿病肾病肾间质纤维化易感性的关系;探讨雷公藤甲素对人小管上皮细胞TSP1/TGFβ1,TLR4/NF-κB信号转导通路的作用。方法:1.用Meta分析的方法,系统检索PubMed、EMBASE、CBM、CSTJ、CJFD及万方数据库以收集关于eNOS 4b/a基因多态性与糖尿病肾病肾间质纤维化易感性关系的病例对照研究。用StataSE12进行统计分析及异质性检验。2.体外培养HK-2细胞,观察低、中和高浓度的雷公藤甲素对TSP1/TGF-β1,TLR4/NF-κB信号转导通路的作用。采用Real Time PCR检测mRNA表达,采用Western blot检测蛋白表达。培养上清液中总TGF-β1和活性TGF-β1检测采用ELISA法。结果:1、本Meta分析纳入26篇糖尿病肾病对比糖尿病的研究及15篇糖尿病肾病对比健康人群(非糖尿病人群)的研究,病例数/对照数分别为6144/4900、2134/2348。纳入研究异质性中度。Meta分析结果显示eNOS 4b/a与糖尿病肾病肾间质纤维化患病风险有显著的相关性。敏感性分析(剔除不满足Hardy-Weinberg平衡定律的研究)检测结果无显著变化。与糖尿病患者相比,等位基因模型显示在总人群(OR=1.26,95%CI:1.10-1.45)、亚洲人种(OR=1.51,95%CI:1.13-2.01)及2型糖尿病患者(OR=1.29,95%CI:1.09-1.54)中有显著相关性。仅在显性模型中漏斗图及Egger检验提示存在发表偏倚(P=0.024)。2、体外研究表明,与各自的阴性对照组比较,阳性组的TSP1/TGF-β1,TLR4/NF-kB的mRNA和蛋白表达均增加。与各自的阳性对照组比较,中/高浓度雷公藤甲素明显下调了TSP1/TGF-β1的mRNA和蛋白表达;也明显下调了TLR4/NF-kB的mRNA和蛋白表达。中/高浓度雷公藤甲素干预后,总的TGF-β1含量和活性TGF-β1含量均明显减少。结论:1、Meta分析的研究表明,eNOS 4b/a基因多态性与糖尿病肾病肾间质纤维化易感性相关,尤其是在亚洲人种,但是还需要设计更严谨的前瞻性的研究以获得更可靠的证据。2、体外研究表明,雷公藤甲素能够下调人肾小管上皮细胞TSP1/TGF-β1的mRNA和蛋白表达,抑制TSP1/TGF-β1通路活化,抑制活性TGF-β1分泌;雷公藤甲素能够下调人肾小管上皮细胞TLR4/NF-kB的mRNA和蛋白表达,抑制TLR4/NF-kB通路的活化。
[Abstract]:Background: the common pathological changes of various chronic renal diseases are tubulointerstitial fibrosis, which is an important cause of end-stage renal failure. "chronic kidney disease-renal interstitial fibrosis" has become the focus of attention in the field of kidney disease in recent years. Interstitial fibrosis in chronic kidney disease is associated with some gene polymorphisms. Activation of multiple signal transduction pathways mediates the development of renal interstitial fibrosis in chronic kidney disease. Triptolide is the main active component of Tripterygium wilfordii. Objective: to investigate the relationship between the polymorphism of endothelial nitric oxide synthase (Enos) 4b / a gene and the susceptibility to renal interstitial fibrosis in diabetic nephropathy. To investigate the effect of triptolide on the signal transduction pathway of TSP1/TGF 尾 1 / TLR 4 / NF- 魏 B in human tubular epithelial cells. In order to collect the case-control study on the relationship between the polymorphism of eNOS 4b / a gene and the susceptibility to renal interstitial fibrosis of diabetic nephropathy, we searched the database of PubMedus EMBASEN CSTJ JFD and Wanfang systematically. The statistical analysis of StataSE12 and heterogeneity test. 2. HK-2 cells were cultured in vitro and observed to be low. The effect of triptolide on TSP1 / TGF- 尾 1 TLR4 / NF- 魏 B signal transduction pathway was neutralized. The expression of mRNA was detected by Real Time PCR. Western blot was used to detect protein expression. Total TGF- 尾 1 and activity TGF- 尾 1 in culture supernatant were detected by ELISA method. Results: 1. This Meta analysis included 26 studies of diabetic nephropathy compared with diabetes mellitus and 15 cases of diabetic nephropathy compared with healthy population (non-diabetic nephropathy). The study of diabetes mellitus, The number of cases / controls were 6144 / 4900 / 2134 / 2348 respectively. The results of meta-analysis showed that eNOS 4b / a was significantly correlated with the risk of renal interstitial fibrosis in diabetic nephropathy. There was no significant change in the results of the study. The allelic model showed a significant correlation between OR1. 2695 CI: 1.10-1.45, the Asian species OR1. 51 / 95 CI: 1.13-2.01and type 2 diabetes patients (OR1. 2995 CIW 1.09-1.54). Only in the dominant model and Egger tests indicated the presence of publication bias P0. 024. 2. In vitro studies showed that there was a difference between the two groups. The expression of mRNA and protein in TSP1 / TGF- 尾 1 + TLR4 / NF-kB was increased. Compared with the positive control group, the mRNA and protein expression of TSP1 / TGF- 尾 1 was significantly down-regulated by triptolide. The expression of mRNA and protein in TLR4/NF-kB was also significantly down-regulated. After the intervention of middle / high concentration of triptolide, The total TGF- 尾 1 content and the activity TGF- 尾 1 content were significantly decreased. Conclusion the study of Meta-analysis shows that the polymorphism of Enos 4b / a gene is associated with the susceptibility to renal interstitial fibrosis of diabetic nephropathy, especially in Asian population. In vitro, triptolide can down-regulate the expression of mRNA and protein in TSP1 / TGF- 尾 1, inhibit the activation of TSP1 / TGF- 尾 1 pathway and inhibit the secretion of active TGF- 尾 1. Triptolide can down-regulate the expression of mRNA and protein in TLR4/NF-kB of human renal tubular epithelial cells and inhibit the activation of TLR4/NF-kB pathway.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R692

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