本文關(guān)鍵詞：TWEAK促進(jìn)人肝星狀細(xì)胞遷移及其機(jī)制探討　出處：《南京醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： TWEAK 肝星狀細(xì)胞 遷移 MMPs 信號通路

【摘要】：背景:肝纖維化是多種慢性肝病進(jìn)展至肝硬化的中間過程,它的各種并發(fā)癥嚴(yán)重的影響著慢性肝病病人的生活質(zhì)量及預(yù)后。目前為止,肝纖維化的治療仍沒有效果確切的藥物。肝星狀細(xì)胞(hepatic stellate cells,HSCs)的活化是肝纖維化發(fā)生發(fā)展過程的核心環(huán)節(jié),其活化后合成大量細(xì)胞外基質(zhì),為肝硬化假小葉的形成創(chuàng)造條件�；诖�,活化的HSCs自然成為逆轉(zhuǎn)肝纖維化的重要靶點(diǎn)。腫瘤壞死因子樣弱凋亡誘導(dǎo)因子(Tumor necrosis factor-like weak inducer of apoptosis,TWEAK)是一個(gè)多功能的細(xì)胞因子,有促炎、血管再生及調(diào)節(jié)細(xì)胞生長、發(fā)育及凋亡的作用。在肝再生時(shí),已有研究表明TWEAK在肝臟中的主要功能是促進(jìn)肝臟原始細(xì)胞的增值,而TWEAK在肝纖維化的發(fā)生發(fā)展過程中所起的作用仍沒有被探討。目的:本研究通過觀察TWEAK對人肝星狀細(xì)胞株--LX-2細(xì)胞增殖、細(xì)胞周期、細(xì)胞凋亡、遷移能力的影響,并探討其中的機(jī)制,以期為肝纖維的治療提供分子靶點(diǎn)依據(jù)。方法:用不同濃度TWEAK培養(yǎng)LX-2細(xì)胞24h或48h,采用CCK-8法檢測TWEAK對LX-2細(xì)胞增殖的影響,流式細(xì)胞術(shù)檢測細(xì)胞周期和細(xì)胞凋亡,Transwell小室檢測LX-2細(xì)胞遷移能力。實(shí)時(shí)定量多聚酶鏈?zhǔn)椒磻?yīng)和蛋白印記用于檢測基質(zhì)金屬蛋白酶(matrixmetalloproteinases,MMPs)、α-SMA、vimentin和desmin的表達(dá),酶聯(lián)免疫吸附試驗(yàn)用于檢測MMPs的活性。運(yùn)用siRNA技術(shù)對LX-2中的MMP9和p65的表達(dá)進(jìn)行干擾。運(yùn)用鬼筆環(huán)肽染色對LX-2的形態(tài)進(jìn)行觀察。結(jié)果:與對照組相比,20ng/mL、40ng/mL和100ng/mLTWEAK都能明顯的增強(qiáng)LX-2細(xì)胞的遷移能力,且100ng/mL較40ng/mLTWEAK作用更強(qiáng);40 ng/mL、100 ng/mL TWEAK對LX-2細(xì)胞的增殖、周期及凋亡無明顯影響。TWEAK明顯增加了 MMP7、MMP8、MMP9和MMP13的表達(dá),并且使MMP9的活性增強(qiáng),使用MMP9 siRNA將MMP9干擾之后,減弱了 TWEAK促進(jìn)的LX-2細(xì)胞的遷移;進(jìn)一步研究發(fā)現(xiàn),TWEAK通過活化經(jīng)典的NF-κB信號通路進(jìn)而增加MMP9的表達(dá)。同時(shí)發(fā)現(xiàn),TWEAK增加了 LX-2細(xì)胞中活化肝星狀細(xì)胞的分子標(biāo)志α-SMA、vimentin和desmin的表達(dá),并且改變了 LX-2的細(xì)胞形態(tài)。結(jié)論:TWEAK通過NF-κB/MMP9信號通路增強(qiáng)LX-2細(xì)胞的遷移;TWEAK能夠促進(jìn)LX-2細(xì)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- 偽). 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, 偽 -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 偽 -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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R575.2

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