支架蛋白JLP在单侧输尿管梗阻小鼠肾间质纤维化中的作用及机制探讨

发布时间：2018-03-12 12:47

本文选题：α-平滑肌肌动蛋白　切入点：输尿管梗阻　出处：《武汉大学》2016年博士论文　论文类型：学位论文

【摘要】：肾间质纤维化是多种进展性肾脏疾病终末阶段共同的病理表现,其病理特征为：肾间质炎性细胞浸润、聚集,肌成纤维细胞(MyoF)增殖、活化和进行性的细胞外基质(ECM)积聚等。JNK相关的亮氨酸拉链蛋白JLP是一种介导生物信号转导及细胞内分子囊泡转运的重要支架蛋白。JLP在小鼠的多种器官组织中被发现,如脑、肺、脾脏、睾丸等。本研究首次探讨了JLP基因缺失对小鼠单侧输尿管梗阻肾病模型肾间质纤维化的影响及其可能机制。第一部分支架蛋白JLP基因缺失对单侧输尿管梗阻小鼠肾间质纤维化的影响目的观察支架蛋白JLP基因缺失对单侧输尿管梗阻(UUO)小鼠肾间质纤维化的影响,探讨JLP在梗阻性肾病肾纤维化形成中的作用及可能机制。方法将jlp野生型jlp+/+)小鼠和jlp敲除(jlp-/-)小鼠分为4组：jlp+/+小鼠假手术组(jlp+/+-Sham)、jlp-/-小鼠假手术组(jlp-/--Sham)、jlp-/-小鼠模型组(jlp-/--UUO)和jlp-/-小鼠模型组(jlp-/--UUO),分别于术后7d和14d处死动物。Masson染色观察肾间质纤维化；免疫荧光、免疫组化及Western印迹观察JLP在正常jlp+/+小鼠肾组织表达与分布；免疫组化法检测肾组织α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(COL-Ⅰ)、Ⅲ型胶原(COL-Ⅲ)表达；Western印迹法检测肾组织α-SMA、COL-Ⅰ、COL-Ⅲ蛋白表达。结果jlp+1+小鼠肾组织表达JLP,主要分布于肾小管。Masson染色显示,与jlp+/+-UUO组比较,jlp-/--UUO组肾间质胶原纤维明显增加,纤维化程度明显加重(P0.05)；免疫组化显示,与jlp-/--UUO组比较,jlp-/--UUO组肾皮质α-SMA、 COL-Ⅰ和COL-Ⅲ表达明显升高(均P0.05);Western印迹显示,jlp-/--UUO组α-SMA、COL-Ⅰ和COL-Ⅲ蛋白表达明显升高(均P0.05)。结论支架蛋白JLP可抑制肌成纤维细胞的生成,在拮抗肾纤维化的形成中起到重要作用。第二部分支架蛋白JLP基因缺失对单侧输尿管梗阻小鼠肾间质纤维化炎症因子表达的影响目的观察支架蛋白JLP基因缺失对UUO小鼠肾间质巨噬细胞聚集、炎症因子释放的影响,探讨JLP在梗阻性肾病肾纤维化形成中的作用。方法将ilp+/+小鼠和jlp-/-小鼠分为4组：jlp+/+-Sham组、jlp-/--Sham组、jlp+/+-UUO组和jlp-/--UUO组,于术后7d处死动物。实时荧光定量PCR检测肾组织肿瘤坏死因子-α(TNF-α)、白细胞介素-1p(IL-1p)和单核细胞趋化蛋白-1(MCP-1)mRNA的表达；免疫组化法检测肾组织巨噬细胞标志物CD68、炎症因子TNF-α、IL-1β和MCP-1的表达；Western印迹检测肾组织TNF-α、IL-1β和MCP-1蛋白的表达。结果实时荧光定量PCR显示,jlp/-/-UUO组TNF-α、IL-1β和MCP-1mRNA表达水平明显升高,与jlp+/+-UUO组比较,差异有统计学意义(均P0.05)；免疫组化显示,与jlp+/+-UUO组比较,jlp-/--UUO组肾皮质CD68、TNF-α、IL-1β和MCP-1表达明显升高(均P0.05)；同时,Western印迹显示,jlp-/--UUO组TNF-α、IL-1β和MCP-1蛋白表达明显升高(均P0.05)结论支架蛋白JLP在抑制梗阻性肾病肾间质巨噬细胞聚集和炎症因子的释放中起到重要作用。第三部分支架蛋白,ILP基因缺失对单侧输尿管梗阻小鼠肾脏TGF-β1/Smad信号通路及细胞凋亡的影响目的观察支架蛋白JLP基因缺失对UUO小鼠肾脏TGF-β1/Smad及肾小管间质细胞凋亡的影响,探讨JLP在梗阻性肾病肾纤维化形成中可能机制。方法将jlp+/+小鼠和jlp-/-小鼠分为4组：jlp+/+-Sham组、jlp-/--Sham组、jlp+/+-UUO组和jlp-/--UUO组,分别于术后7d和14d处死动物。实时荧光定量PCR检测肾组织转化生长因子β1(TGF-β1)mRNA的表达；免疫组化法检测肾组织TGF-β1的表达；Western印迹检测肾组织TGF-β1、p-Smad2、p-Smad3及p-P65蛋白的表达；TUNEL法检测肾脏间质细胞凋亡。结果实时荧光定量PCR显示,jlp-/--UUO组TGF-β1mRNA表达水平明显升高,与jlp+/+-UUO组比较,差异有统计学意义(P0.05)；免疫组化显示,与jlp+/+-UUO组比较,jlp-/--UUO组肾皮质TGF-β1表达明显升高(P0.05)；同时,Western印迹显示,jlp-/--UUO组TGF-β1、p-Smad2、p-Smad3及p-P65蛋白表达明显升高(均P0.05)；TUNEL显示,与jlp+/+-UUO组比较,jlp-/--UUO组肾脏间质细胞凋亡率明显升高(P0.05)。结论支架蛋白JLP可抑制TGF-β1/Smad信号通路及NF-κB信号通路的激活、并减少细胞凋亡,在拮抗肾纤维化的形成中起重要作用。
[Abstract]:Renal interstitial fibrosis is a pathologic variety of progressive renal disease, end stage common manifestations, the pathological features of renal interstitial infiltration of inflammatory cells, aggregation, fibroblast (MyoF) proliferation, activation and extracellular matrix (ECM) accumulation of.JNK related leucine zipper protein JLP is a kind of important biological scaffold protein.JLP mediated signal transduction and cellular vesicular transport has been found in a variety of organs and tissues in mice, such as brain, lung, spleen, testis and so on. This study is the first to investigate the effects of renal JLP gene deletion on model mice with unilateral ureteral obstruction renal interstitial fibrosis and its possible mechanism the first part. The scaffold protein JLP gene deletion in mice with unilateral ureteral obstruction on renal interstitial fibrosis Objective To observe the effects of scaffold protein JLP gene deletion in unilateral ureteral obstruction (UUO) mice renal interstitial fibrosis, explore JLP In renal fibrosis in obstructive nephropathy in the formation of the effects and the possible mechanism. Methods: JLP wild type jlp+/+) mice and JLP knockout (jlp-/-) mice were divided into 4 groups: sham operation group jlp+/+ mice (jlp+/+-Sham mice), jlp-/- sham operation group (jlp-/--Sham), jlp-/- model group (jlp-/--UUO) and jlp-/- mice model group (jlp-/--UUO), respectively after 7d and 14d were sacrificed animal.Masson staining to observe the renal interstitial fibrosis; immunofluorescence, immunohistochemistry and Western blotting to observe the expression and distribution of JLP in renal tissue of normal jlp+/+ mice; immunohistochemistry in renal tissue of alpha smooth muscle actin (alpha -SMA), collagen (COL- I), collagen type III (COL- III) expression in renal tissue was detected by Western blotting; alpha -SMA, COL- 1, COL- expression protein. Results the expression of JLP in renal tissue of jlp+1+ mice, mainly in renal tubular.Masson staining showed that compared with jlp+/+-UUO group, jlp-/--U UO group of renal interstitial collagen fibers increased significantly, the degree of fibrosis was significantly increased (P0.05); immunohistochemistry showed that, compared with jlp-/--UUO group, jlp-/--UUO group of renal cortical alpha -SMA, expression of COL- I and COL- III were significantly increased (P0.05); Western blot showed that jlp-/--UUO group, alpha -SMA, COL- I and COL- III protein expression increased significantly (P0.05). Conclusion the scaffold protein JLP can inhibit the generation of muscle fiber cells, play an important role in the formation of anti renal fibrosis. Some of the scaffold protein JLP gene deletion second on expression of inflammatory factor of fiber in mice with unilateral ureteral obstruction kidney to observe the scaffold protein JLP gene deletion in UUO mice renal interstitial macrophages, inflammatory factors release, formation of JLP in renal fibrosis in obstructive nephropathy in rats. Methods ilp+/+ mice and jlp-/- mice were divided into 4 groups: jlp+/+ -Sham group, jlp-/--Sha M group, jlp+/+-UUO group and jlp-/--UUO group, sacrificed on 7d animal kidney tissue. Real time fluorescence quantitative PCR detection of tumor necrosis factor alpha (TNF- alpha), interleukin -1p (IL-1p) and monocyte chemoattractant protein -1 (MCP-1) mRNA expression; immunohistochemistry in renal tissue of giant macrophage marker CD68, inflammatory factor TNF- alpha, IL-1 beta and MCP-1 expression; Western blot detection of TNF- expression in renal tissue of alpha, IL-1 beta and MCP-1 protein. Results of real-time PCR showed that jlp/-/-UUO group TNF- alpha, IL-1 beta and MCP-1mRNA expression levels were significantly increased, compared with jlp+/+-UUO group, the difference was statistically significant (P0.05); immunohistochemistry showed that, compared with jlp+/+-UUO group, jlp-/--UUO group, renal cortex CD68, TNF- alpha, IL-1 beta and MCP-1 expression increased significantly (P0.05); at the same time, Western blotting showed that the jlp-/--UUO group of TNF- alpha, IL-1 beta and MCP-1 protein expression increased significantly (P0.05) conclusion Play an important role in the inhibition of the scaffold protein JLP in obstructive nephropathy renal interstitial macrophage accumulation and release of inflammatory factors. The third part scaffold protein, the effect of ILP gene deletion on mouse kidney with unilateral ureteral obstruction TGF- beta 1/Smad signaling pathway and apoptosis to observe support protein JLP gene deletion effect on UUO mouse kidney TGF- beta 1/Smad and tubulointerstitial cell apoptosis, explore the formation of JLP in renal fibrosis in obstructive nephropathy in the possible mechanism. Methods: jlp+/+ mice and jlp-/- mice were divided into 4 groups: jlp+/+-Sham group, jlp-/--Sham group, jlp+/+-UUO group and jlp-/--UUO group, respectively after 7d and 14d were animal. Real time fluorescence quantitative PCR detection of renal tissue transformation growth factor beta 1 (TGF- beta 1) mRNA expression; immunohistochemistry was used to detect the expression of TGF- in renal tissue beta 1; renal tissue Western blot detection of TGF- beta 1, p-Smad2, p-Smad3 and p-P65 protein The expression of TUNEL was detected; renal interstitial cell apoptosis. Results of real-time PCR showed that the expression of jlp-/--UUO in group TGF- beta 1mRNA levels were significantly increased, compared with jlp+/+-UUO group, the difference was statistically significant (P0.05); immunohistochemistry showed that, compared with jlp+/+-UUO group, jlp-/ --UUO group of renal cortical TGF- expression was significantly increased (p 1 P0.05); at the same time, Western blotting showed that jlp-/--UUO group TGF- beta 1, p-Smad2, p-Smad3 and the expression of p-P65 protein increased significantly (P0.05); TUNEL showed that, compared with jlp+/+-UUO group, jlp-/--UUO group of renal interstitial cell apoptosis rate increased significantly (P0.05). Conclusion: the activation of the scaffold protein JLP can inhibit the 1/Smad signaling pathway and TGF- NF- B signaling pathway, and reduce apoptosis, play an important role in the formation of anti renal fibrosis.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R692

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