TWEAK促进人肝星状细胞迁移及其机制探讨

发布时间：2017-12-31 08:32

本文关键词：TWEAK促进人肝星状细胞迁移及其机制探讨　出处：《南京医科大学》2017年硕士论文　论文类型：学位论文

更多相关文章： TWEAK 肝星状细胞 迁移 MMPs 信号通路

【摘要】：背景:肝纤维化是多种慢性肝病进展至肝硬化的中间过程,它的各种并发症严重的影响着慢性肝病病人的生活质量及预后。目前为止,肝纤维化的治疗仍没有效果确切的药物。肝星状细胞(hepatic stellate cells,HSCs)的活化是肝纤维化发生发展过程的核心环节,其活化后合成大量细胞外基质,为肝硬化假小叶的形成创造条件。基于此,活化的HSCs自然成为逆转肝纤维化的重要靶点。肿瘤坏死因子样弱凋亡诱导因子(Tumor necrosis factor-like weak inducer of apoptosis,TWEAK)是一个多功能的细胞因子,有促炎、血管再生及调节细胞生长、发育及凋亡的作用。在肝再生时,已有研究表明TWEAK在肝脏中的主要功能是促进肝脏原始细胞的增值,而TWEAK在肝纤维化的发生发展过程中所起的作用仍没有被探讨。目的:本研究通过观察TWEAK对人肝星状细胞株--LX-2细胞增殖、细胞周期、细胞凋亡、迁移能力的影响,并探讨其中的机制,以期为肝纤维的治疗提供分子靶点依据。方法:用不同浓度TWEAK培养LX-2细胞24h或48h,采用CCK-8法检测TWEAK对LX-2细胞增殖的影响,流式细胞术检测细胞周期和细胞凋亡,Transwell小室检测LX-2细胞迁移能力。实时定量多聚酶链式反应和蛋白印记用于检测基质金属蛋白酶(matrixmetalloproteinases,MMPs)、α-SMA、vimentin和desmin的表达,酶联免疫吸附试验用于检测MMPs的活性。运用siRNA技术对LX-2中的MMP9和p65的表达进行干扰。运用鬼笔环肽染色对LX-2的形态进行观察。结果:与对照组相比,20ng/mL、40ng/mL和100ng/mLTWEAK都能明显的增强LX-2细胞的迁移能力,且100ng/mL较40ng/mLTWEAK作用更强;40 ng/mL、100 ng/mL TWEAK对LX-2细胞的增殖、周期及凋亡无明显影响。TWEAK明显增加了 MMP7、MMP8、MMP9和MMP13的表达,并且使MMP9的活性增强,使用MMP9 siRNA将MMP9干扰之后,减弱了 TWEAK促进的LX-2细胞的迁移;进一步研究发现,TWEAK通过活化经典的NF-κB信号通路进而增加MMP9的表达。同时发现,TWEAK增加了 LX-2细胞中活化肝星状细胞的分子标志α-SMA、vimentin和desmin的表达,并且改变了 LX-2的细胞形态。结论:TWEAK通过NF-κB/MMP9信号通路增强LX-2细胞的迁移;TWEAK能够促进LX-2细胞活化。
[Abstract]:Background: liver fibrosis is an intermediate process from chronic liver disease to liver cirrhosis. Its complications seriously affect the quality of life and prognosis of patients with chronic liver disease. The treatment of hepatic fibrosis still has no effective drug. Hepatic stellate cell hepatic stellate cells. The activation of HSCs is the key link in the development of hepatic fibrosis. After activation, a large number of extracellular matrix is synthesized, which creates the conditions for the formation of liver cirrhosis pseudolobules. Activated HSCs has naturally become an important target for reversing liver fibrosis. Tumor Necrosis Factor-Like weak apoptosis Induction Factor (TNF- 伪). Tumor necrosis factor-like weak inducer of apoptosis. TWEAK is a multifunctional cytokine that promotes inflammation, vascular regeneration and regulates cell growth, development and apoptosis. Studies have shown that the main function of TWEAK in the liver is to promote the proliferation of liver primordial cells. However, the role of TWEAK in the development of hepatic fibrosis has not been explored. Objective: to investigate the effect of TWEAK on the proliferation and cell cycle of human hepatic stellate cell line LX-2. The effect of apoptosis and migration ability and the mechanism were discussed in order to provide molecular target for the treatment of liver fiber. Methods: LX-2 cells were cultured with different concentrations of TWEAK for 24 hours or 48 hours. CCK-8 assay was used to detect the effect of TWEAK on the proliferation of LX-2 cells. Flow cytometry was used to detect cell cycle and apoptosis. Transwell chambers for the detection of migration in LX-2 cells. Real-time quantitative polymerase chain reaction and protein imprinting are used to detect matrix metalloproteinases (MMPs). Matrixmetalloproteinases. Expression of MMPsM, 伪 -SMAV vimentin and desmin. The enzyme linked immunosorbent assay (Elisa) was used to detect the activity of MMPs. The expression of MMP9 and p65 in LX-2 was interfered by siRNA technique. The morphology of LX-2 was detected by using Gypanopeptide staining. Observations. Results:. Compared with the control group. Both 20ng / mLL 40ng / mL and 100ng / mLTWEAK significantly enhanced the migration ability of LX-2 cells. The effect of 100ng / mL was stronger than that of 40ng / mL LTWEAK. 40 ng / mL 100 ng/mL TWEAK had no effect on the proliferation, cell cycle and apoptosis of LX-2 cells. TWEAK significantly increased MMP7 and MMP8. The expression of MMP9 and MMP13, and increased the activity of MMP9, after using MMP9 siRNA to interfere with MMP9. The migration of LX-2 cells stimulated by TWEAK was attenuated. It was further found that TWEAK increased the expression of MMP9 by activating the classical NF- 魏 B signaling pathway. TWEAK increased the expression of 伪 -SMAV vimentin and desmin in LX-2 cells. It also changed the cell morphology of LX-2. Conclusion: TWEAK enhances the migration of LX-2 cells through NF- 魏 B / MMP9 signaling pathway. TWEAK can promote the activation of LX-2 cells.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2

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