环氧合酶-2在TNF-α诱导的人晶状体上皮细胞增殖和转分化中的作用及其机制研究

发布时间：2018-04-06 23:06

本文选题：肿瘤坏死因子-α　切入点：人晶状体上皮细胞　出处：《浙江大学》2016年博士论文

【摘要】：背景与目的后发性白内障(又称后囊膜浑浊,posterior capsular opacification, PCO)是目前白内障术后最主要的并发症,其主要的病理机制是术后残留的晶状体上皮在活化或升高的细胞因子等作用下发生细胞增殖、迁移和上皮向间充质转分化等。研究发现肿瘤坏死因子-α (TNF-α)是白内障术后房水中存在的一种重要的炎症因子,被认为和PCO的形成有关。另外有研究表明环氧合酶-2(Cyclooxygenase-2, COX-2)抑制剂在体内实验和体外模拟实验中均可以有效地抑制PCO的发生,但其药理作用的分子机制仍不明确。因此本学位论文旨在通过体外培养的人晶状体上皮细胞模型,探究TNF-α对COX-2的表达调控机制,及COX-2在TNF-α诱导的人晶状体上皮细胞增殖和转分化中的作用及其可能的机制。方法体外培养的人晶状体上皮细胞(hLEC-B3)分为两组,即10 ng/ml TNF-α刺激组和无TNF-α刺激的对照组,在刺激后6 h、24 h、48 h,用实时定量PCR和Western Blot检测COX-2 mRNA和蛋白的表达水平。同时,经TNF-a刺激后24 h,用定量PCR和Western Blot检测细胞增殖和EMT的标志物细胞周期蛋白D1(Cyclin D1)和波形蛋白(Vimentin) mRNA及蛋白表达水平,并用Cell Count Kit-8(CCK-8)试剂盒和Transwell迁移小室实验分别检测细胞增殖和迁移能力。在光学显微镜观察细胞形态变化。在TNF-α刺激前加入溴芬酸钠24 h以阻断COX-2,然后检测细胞增殖和EMT的改变。观察hLEC-B3细胞经10 ng/ml TNF-α刺激时间梯度效应,用Western Blot检测JNK磷酸化水平的改变以及转录因子c-Jun的活化水平,提取细胞核蛋白的检测和细胞免疫荧光的方法来观察p-c-Jun的入核情况。预先加入JNK抑制剂SP600125或转染c-Jun siRNA后检测COX-2表达的改变,以鉴定JNK信号通路对COX-2表达的调控作用,并利用染色质免疫共沉淀(ChIP)验证p-c-Jun与COX-2启动子区DNA序列的直接相互作用。为进一步探索COX-2可能介导的机制,通过Western Blot及双荧光素酶报告基因系统的方法来研究TNF-α对GSK-3β/β-catenin通路以及β/-catenin转录活性的影响,并通过阻断COX-2或JNK通路以探究JNK/COX-2在GSK-3β/β-catenin通路活化中的作用。结果TNF-α可以诱导hLEC-B3细胞COX-2的高表达以及Cyclin D1和Vimentin表达的升高,并促进细胞增殖、迁移和纤维化形态改变。预先加入澳芬酸钠阻断COX-2可以抑制TNF-α诱导的Cyclin D1和Vimentin的高表达及细胞增殖和EMT。TNF-α通过激活JNK/p-c-Jun信号通路调控COX-2的表达,ChIP-qPCR结果显示p-c-Jun与COX-2启动子区DNA序列具有直接相互作用。另外,TNF-α可以激活GSK-3β/β-catenin通路并提高β-catenin转录活性。药物阻断COX-2或JNK信号通路均可以抑制TNF-a活化的GSK-3β/β-catenin通路和β-catenin转录活性。转染β-catenin siRNA可减弱了溴芬酸钠对TNF-α诱导的Cyclin D1和Vimentin高表达的抑制效应。结论TNF-α通过JNK信号转导通路调控COX-2的表达。JNK/COX-2可能通过介导GSK-3β/β-catenin通路,在TNF-α诱导的hLEC-B3细胞增殖和EMT中发挥了重要作用。
[Abstract]:Background and objective after cataract (also known as posterior capsular opacification, posterior capsular opacification, PCO) is the main complication after cataract surgery, the main pathological mechanism of postoperative residual lens epithelial cell proliferation occurs in the activation or elevated cytokines induced by migration and epithelial to mesenchymal. The study found that differentiation. Tumor necrosis factor alpha (TNF- alpha) is an important inflammatory factor in aqueous humor after cataract surgery, is believed to form and PCO. Other studies have shown that cyclooxygenase -2 (Cyclooxygenase-2, COX-2) can effectively inhibit the occurrence of inhibitors of PCO in vivo and in vitro in the simulation experiment, but the molecular mechanism of pharmacological action is still not clear. Therefore, this dissertation aims to model human lens epithelial cells by in vitro culture, explore the mechanism of expression and regulation of TNF- alpha on COX-2 And the mechanism of the proliferation of human lens epithelial cells and COX-2 in TNF- induced transdifferentiation and the role and possible. Human lens epithelial cells in vitro (hLEC-B3) were divided into two groups, namely control group 10 ng/ml TNF- stimulation group and TNF- stimulation, after stimulation of 6 h, 24 h 48, H expression by real-time quantitative PCR and Western Blot detection of COX-2 mRNA and protein. At the same time, after stimulated by TNF-a 24 h, Western Blot and PCR signs of quantitative detection of cell proliferation and cell cycle protein EMT D1 (Cyclin D1) and vimentin (Vimentin) expression of mRNA and protein, and Cell Count Kit-8 (CCK-8) Transwell kit and cell migration experiment were used to detect the proliferation and migration of cells. Morphological changes were observed under optical microscope. The TNF- stimulation before adding bromfenac sodium 24 h to block COX-2, then detected the proliferation and EMT cell changes. HLEC-B3 cells were observed after 10 ng/ml TNF- stimulation time gradient effect, using Western Blot to detect the change of JNK phosphorylation level and the activation level of the transcription factor c-Jun, extraction method and immunofluorescence to observe the nuclear protein p-c-Jun into the nucleus. The pre accession to the JNK inhibitor SP600125 or c-Jun siRNA to detect the expression of COX-2 after transfection the change of role in identification of JNK signaling pathway on the expression of COX-2, and the use of chromatin immunoprecipitation (ChIP) validation of p-c-Jun with COX-2 promoter direct interaction promoter sequence of DNA. To further explore the mechanism of COX-2 may be mediated by Western, Blot and dual luciferase reporter system to study TNF- the effect of alpha GSK-3 beta beta beta / -catenin pathway and transcriptional activity of /-catenin, and by blocking COX-2 or JNK pathway to explore the JNK/COX-2 in the GSK-3 beta / beta -catenin pathway. In the interactions of the TNF- alpha hLEC-B3 cells can be induced by the high expression of COX-2 and Cyclin expression of D1 and Vimentin increased, and promote cell proliferation, migration and fibrosis change form. Pretreatment with acid sodium can inhibit offen blocked COX-2 induced by TNF- Cyclin D1 and the high expression of Vimentin and cell proliferation and EMT.TNF- expression by alpha the activation of JNK/p-c-Jun signal transduction pathway of COX-2, p-c-Jun and ChIP-qPCR showed that COX-2 promoter DNA sequence has a direct interaction. In addition, TNF- can activate GSK-3 alpha beta / beta -catenin pathway and increased beta -catenin transcription activity. Drugs blocking COX-2 or JNK signaling pathway can inhibit the activation of TNF-a GSK-3 pathway and -catenin / beta beta beta the transcriptional activity of -catenin. Transfection beta -catenin siRNA can weaken the inhibitory effect of high expression of bromfenac sodium on TNF- induced Cyclin D1 and Vimentin. Conclusion TNF- alpha by JN K signal transduction pathway regulates the expression of COX-2..JNK/COX-2 may play an important role in TNF- induced hLEC-B3 cell proliferation and EMT by mediating the GSK-3 beta / -catenin pathway.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R776.1

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