TWEAK与增殖性糖尿病视网膜病变相关性及促细胞增殖作用的研究

发布时间：2018-03-07 13:01

本文选题：TWEAK　切入点：增殖性糖尿病视网膜病变(PDR)　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:为了证实TWEAK和Fn14在增殖性糖尿病视网膜病变中上调,并有促使ARPE-19细胞增殖和胶原合成的作用。研究背景:糖尿病视网膜病变严重危害了糖尿病患者的视力,是其视力丧失的主要原因,是糖尿病危害性最大的并发症之一。持续的高血糖引起视网膜微血管的慢性病变,促进糖尿病视网膜病变病理过程的进展。临床上根据糖尿病视网膜病变(DR)的分级标准,出现视网膜新生血管或玻璃体出血和(或)视网膜前出血即发展为增殖性糖尿病视网膜病变(PDR)。PDR引起反复玻璃体出血,视网膜和玻璃体纤维化,视神经萎缩以及视网膜脱离,最终可导致患者视力严重受损。PDR最显著的特征就是视网膜新生血管的形成,各种血管因子、细胞因子和炎症因子均参与这一过程。以往的重点在于各类血管内皮生长因子和其它具有促血管生成作用的因子的研究。近年来,免疫系统的紊乱和功能失衡以及血管增殖的激活也被认为参与了PDR的病理过程。另外有研究表明,在PDR增殖过程中有多种细胞参与,如成纤维细胞、RPE细胞、胶原细胞等,因RPE细胞参与血-视网膜外屏障的构成,所以RPE细胞的变化在PDR进展中尤为重要。最近肿瘤坏死因子超家族中的新成员被发现具有调节炎症和细胞增殖、促新生血管形成的作用。特别是肿瘤坏死因子样的凋亡弱诱导剂(TWEAK)具有活跃的生物学特性,它通过结合纤维母细胞生长因子诱导分子14(Fn14)激活NF-κB信号通路,调节细胞的存活和增殖,诱导释放Cathepsin B引起细胞死亡,提高前炎症分子MMP9、ICAM1和IL6的表达,而这些细胞因子均已被证实参与了PDR的病理过程。有报道称,在PDR病人玻璃体中有TWEAK和Fn14的基因表达。大量证据表明TWEAK有可能通过TWEAK/Fn14信号通路参与调控PDR的发展。本研究通过检测PDR病人玻璃体中TWEAK和Fn14的表达情况,了解TWEAK和Fn14与PDR的相关性。为确认TWEAK表达与PDR临床病理特征之间的关系,我们进一步研究了视网膜细胞过表达TWEAK后,能够提高该细胞增殖和胶原合成的能力。方法:玻璃体样本的采集通过玻璃体手术分别获得16个PDR病人和21个非PDR的2型糖尿病病人的玻璃体样本。ELISA法检测玻璃体液中TWEAK和Fn14含量通过ELISA法检测两组病人玻璃体液中TWEAK和Fn14的含量。Westernblot法分析玻璃体液中TWEAK和Fn14蛋白的表达量两组病人玻璃体液中TWEAK和Fn14蛋白的表达水平通过与GAPDH带的灰度百分比表示。目的基因的获取从293T细胞获取人的总DNA,通过PCR技术获得TWEAK扩展片段。ARPE-19细胞培养和TWEAK过表达将ARPE-19细胞进行贴壁培养。细胞融合大于90%时,以1:3的比例分代。在ARPE-19细胞中过表达TWEAK。将TWEAK编码序列克隆入pc DNA3.1(+)载体。将TWEAK-pc DNA3.1(+)和对照质粒分别转染入ARPE-19细胞。在G418液体中筛选并保存。RNA提取和定量RT-PCRRT-PCR操作按照试剂盒说明书进行,GAPDH作为内对照。TWEAK和GAPDH引物由Sangon公司合成。Westernblot法分析细胞中TWEAK蛋白的表达量检测过表达ARPE-19细胞在传代不同阶段,细胞中TWEAK蛋白的表达量。细胞计数检测,MTT检测和群落形成检测细胞计数检测获得细胞生长曲线,MTT法检测细胞活力,集落形成实验检测细胞增殖能力。Westernblot法分析细胞中α-SMA,collagen I和collagen IV蛋白的表达量检测过表达ARPE-19细胞在传代不同阶段,细胞中α-SMA,collagen I和collagen IV蛋白的表达量。统计和分析用Graph Pad Prism软件进行统计分析。数据结果表示为平均值加减标准差。两组间对比采用t-test,多组均数对比采用单因素方差分析。P小于0.05视为有统计学意义。结果:PDR病人玻璃体液中TWEAK和Fn14水平升高21个非PDR的T2DM病人和16个PDR病人的玻璃体样本被收录本研究中。为确定TWEAK和Fn14与PDR的相关性,通过ELISA法检测了两组病人玻璃体液中TWEAK和Fn14的含量的变化,结果显示,PDR病人玻璃体中TWEAK和Fn14含量显著升高。通过Westernblot法分析了两组病人玻璃体中TWEAK和Fn14的蛋白表达水平。结果显示,TWEAK和Fn14在PDR病人玻璃体中的蛋白表达水平也随之显著升高,因此,我们认为TWEAK/Fn14信号通路在PDR病人的玻璃体液中表达上调。稳定的TWEAK过表达ARPE-19细胞系的建立从293T细胞中获取人总DNA,利用PCR技术得到TWEAK编码序列扩展片段,然后被克隆入pc DNA3.1(+)载体,ARPE-19细胞被克隆的载体转染,可以获得稳定表达TWEAK的ARPE-19细胞系。TWEAK过表达促进了细胞增殖和胶原在ARPE-19中的合成为了研究TWEAK在ARPE-19细胞增殖中的作用,我们将ARPE-19(TWEAK过表达)组和ARPE-19(阴性对照)组的细胞生长曲线进行对比,我们发现,TWEAK过表达组细胞群在培育3天和5天时的生长速度明显加快。在培育24和48小时后,过表达组细胞的活力较对照组更强。无论初始培育的细胞数目是多少,TWEAK过表达组细胞形成的集落数目显著多于对照组。因此我们认为TWEAK过表达可以引起ARPE-19细胞的增殖和活力的增强。我们进一步研究了纤维化相关分子在两组细胞中的表达情况,结果显示,过表达组细胞中α-SMA,collagen I和collagen IV的蛋白表达水平显著高于对照组,并在细胞传代过程中表达稳定,提示TWEAK能提高纤维化相关分子在视网膜细胞中的表达。结论:在增殖性糖尿病视网膜病变病人的玻璃体中,TWEAK/Fn14通路表达上调。TWEAK/Fn14通过其相关机制推动PDR的病理发展过程。TWEAK/Fn14通路在视网膜ARPE-19细胞增殖和胶原合成中的发挥调节作用。
[Abstract]:Objective: To observe the TWEAK and Fn14 in proliferative diabetic retinopathy and the up-regulated ARPE-19 cell proliferation and collagen synthesis. Background: diabetic retinopathy, serious harm to the eyesight of patients with diabetes, which is the main reason for the loss of vision, is one of the most harmful complications of diabetes. Persistent hyperglycemia induced chronic pathological changes of retinal microvascular, promote the progression of retinopathy in diabetes. According to the clinical pathology of diabetic retinopathy (DR) grading standards, retinal neovascularization or vitreous body hemorrhage and retinal hemorrhage (or) is developed for proliferative diabetic retinopathy (PDR) caused by.PDR repeated vitreous body hemorrhage, vitreous body and retinal fibrosis, optic nerve atrophy and retinal detachment, ultimately lead to characteristics of patients with severe visual impairment is the most significant.PDR Retinal neovascularization, vascular factors, cytokines and inflammatory factors are involved in this process. The focus is on various types of vascular endothelial growth factor and other angiogenic effect factors. In recent years, the disorder and function of the immune system imbalance of vascular proliferation and activation is also thought to be involved in the pathology the process of PDR. Other studies have shown that there are many cells involved in PDR proliferation, such as fibroblasts, RPE cells, collagen cells, because of the involvement of RPE cells in the blood retinal barrier, so the change of RPE cells in the progression of PDR is particularly important. Recently, a new member of TNF superfamily the regulation was found to have inflammation and cell proliferation, promote angiogenesis. Especially the apoptosis of tumor necrosis factor like weak inducer (TWEAK) has the characteristics of biological active, it A combination of fibroblast growth factor 14 (Fn14) to induce activation of NF- B signaling pathway, regulating cell survival and proliferation, induce the release of Cathepsin B induced cell death, increased proinflammatory molecules MMP9, expression of ICAM1 and IL6, and these cytokines have been of the argument and PDR have reported pathological processes. Said, the expression of TWEAK and Fn14 gene in PDR patients in the vitreous body. Abundant evidence for the development of TWEAK through TWEAK/Fn14 signaling pathway involved in the regulation of PDR. The expression was detected by TWEAK and Fn14 in patients with PDR in the solution of vitreous body, TWEAK and Fn14 and PDR correlation. As to identify the relationship between the expression of TWEAK and PDR the clinical and pathological features, we further study the retinal cells after overexpression of TWEAK can enhance the ability of proliferation and collagen synthesis of the cells. Methods: the samples collected by vitreous body vitreous body surgery Don't get 16 PDR patients and 21 non PDR patients with type 2 diabetes samples detected by.ELISA vitreous body in the vitreous of TWEAK and Fn14 content by content of.Westernblot were detected by ELISA TWEAK and Fn14 glass in two groups of patients in the body fluid analysis of TWEAK and Fn14 protein in the vitreous surface expression of TWEAK and Fn14 protein expression quantity the glass of two groups of patients in body fluids by gray percentage and GAPDH band said. Target gene DNA to obtain the total acquisition from 293T cells,.ARPE-19 cells and obtain extended fragments over expression of TWEAK ARPE-19 cells were cultured by TWEAK PCR. Cell fusion is more than 90%, with the ratio of 1:3 generation. Overexpression of TWEAK. TWEAK encoding sequence was cloned into PC DNA3.1 cells in ARPE-19 (+) vector. The TWEAK-pc (+) DNA3.1 were transfected into ARPE-19 cells and the control plasmid. In G418 liquid screening and preservation .RNA extraction and quantitative RT-PCRRT-PCR operation in accordance with the kit, GAPDH was used.TWEAK and GAPDH primers by Sangon synthesis.Westernblot analysis of TWEAK protein expression in cells detected expression of ARPE-19 cells in different stages of subculture, expression of TWEAK protein in cells. Cell count, MTT assay and colony forming cell detection counting detection of cell growth curve, cell viability was measured by MTT assay, colony formation assay cell proliferation.Westernblot assay cell alpha -SMA expression of collagen I and collagen IV protein detected expression of ARPE-19 cells in different stages of subculture, cells in a -SMA, expression of collagen I and collagen IV protein. Analysis and statistical analysis with Graph Pad Prism software. The data were expressed as mean and standard deviation. The comparison between the two groups by t-test, multi The number of groups were compared with single factor analysis of variance.P less than 0.05 was considered as statistically significant. Results: the levels of serum TWEAK and Fn14 in patients with PDR in the vitreous of 21 T2DM patients and 16 non PDR patients with PDR were included in the study sample of vitreous body. In order to determine the correlation of TWEAK and Fn14 and PDR, display the results ELISA assay was used to determine the changes in the contents of TWEAK and Fn14 in two groups of patients in the vitreous of TWEAK and Fn14 in PDR patients were significantly increased in the vitreous body. Through the Westernblot analysis of the TWEAK and Fn14 of two groups of patients in the vitreous body protein expression level. The results showed that TWEAK and Fn14 in patients with PDR protein expression in the vitreous body also increased significantly, therefore, we believe that the expression of TWEAK/Fn14 signaling pathway in the vitreous of patients with PDR. The stable TWEAK expression ARPE-19 cell line was established to obtain the total DNA from 293T cells, and Using PCR technology TWEAK encoding sequence fragment, then was cloned into the PC vector, DNA3.1 (+) vector transfected ARPE-19 cells were cloned, can obtain the stable expression of ARPE-19 cell line.TWEAK over expression of TWEAK promoted cell proliferation and collagen synthesis in ARPE-19 in order to study the TWEAK in ARPE-19 cell proliferation, we ARPE-19 (expression of TWEAK) and ARPE-19 group (negative control group) the cell growth curves were compared, we found that TWEAK overexpression group group of cells in the cultivation and growth speed of 3 days and 5 days significantly accelerated. In the cultivation of 24 and 48 hours later, cells viability compared with the control group is stronger. No matter the number of initial cell cultivation is the number of TWEAK cells of the colony number was significantly more than the control group. So we think that TWEAK overexpression can lead to enhanced proliferation and viability of ARPE-19 cells. We Further research on the expression of fibrosis related genes in two groups of cells showed that the cells in a -SMA, the expression level of collagen I and collagen IV protein was significantly higher than the control group, and stable expression during passage in cells, suggesting that TWEAK can improve the fiber expression of related molecules in the cells in the retina. Conclusion: the vitreous body in proliferative diabetic retinopathy patients, TWEAK/Fn14 pathway up-regulated.TWEAK/Fn14 expression through its relevant mechanisms to promote the pathological process of.TWEAK/Fn14 pathway in PDR increased proliferation and collagen synthesis play a regulatory role in retinal ARPE-19 cells.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2;R774.1

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