本文選題：TWEAK　切入點(diǎn)：增殖性糖尿病視網(wǎng)膜病變(PDR)　出處：《吉林大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：目的:為了證實(shí)TWEAK和Fn14在增殖性糖尿病視網(wǎng)膜病變中上調(diào),并有促使ARPE-19細(xì)胞增殖和膠原合成的作用。研究背景:糖尿病視網(wǎng)膜病變嚴(yán)重危害了糖尿病患者的視力,是其視力喪失的主要原因,是糖尿病危害性最大的并發(fā)癥之一。持續(xù)的高血糖引起視網(wǎng)膜微血管的慢性病變,促進(jìn)糖尿病視網(wǎng)膜病變病理過(guò)程的進(jìn)展。臨床上根據(jù)糖尿病視網(wǎng)膜病變(DR)的分級(jí)標(biāo)準(zhǔn),出現(xiàn)視網(wǎng)膜新生血管或玻璃體出血和(或)視網(wǎng)膜前出血即發(fā)展為增殖性糖尿病視網(wǎng)膜病變(PDR)。PDR引起反復(fù)玻璃體出血,視網(wǎng)膜和玻璃體纖維化,視神經(jīng)萎縮以及視網(wǎng)膜脫離,最終可導(dǎo)致患者視力嚴(yán)重受損。PDR最顯著的特征就是視網(wǎng)膜新生血管的形成,各種血管因子、細(xì)胞因子和炎癥因子均參與這一過(guò)程。以往的重點(diǎn)在于各類血管內(nèi)皮生長(zhǎng)因子和其它具有促血管生成作用的因子的研究。近年來(lái),免疫系統(tǒng)的紊亂和功能失衡以及血管增殖的激活也被認(rèn)為參與了PDR的病理過(guò)程。另外有研究表明,在PDR增殖過(guò)程中有多種細(xì)胞參與,如成纖維細(xì)胞、RPE細(xì)胞、膠原細(xì)胞等,因RPE細(xì)胞參與血-視網(wǎng)膜外屏障的構(gòu)成,所以RPE細(xì)胞的變化在PDR進(jìn)展中尤為重要。最近腫瘤壞死因子超家族中的新成員被發(fā)現(xiàn)具有調(diào)節(jié)炎癥和細(xì)胞增殖、促新生血管形成的作用。特別是腫瘤壞死因子樣的凋亡弱誘導(dǎo)劑(TWEAK)具有活躍的生物學(xué)特性,它通過(guò)結(jié)合纖維母細(xì)胞生長(zhǎng)因子誘導(dǎo)分子14(Fn14)激活NF-κB信號(hào)通路,調(diào)節(jié)細(xì)胞的存活和增殖,誘導(dǎo)釋放Cathepsin B引起細(xì)胞死亡,提高前炎癥分子MMP9、ICAM1和IL6的表達(dá),而這些細(xì)胞因子均已被證實(shí)參與了PDR的病理過(guò)程。有報(bào)道稱,在PDR病人玻璃體中有TWEAK和Fn14的基因表達(dá)。大量證據(jù)表明TWEAK有可能通過(guò)TWEAK/Fn14信號(hào)通路參與調(diào)控PDR的發(fā)展。本研究通過(guò)檢測(cè)PDR病人玻璃體中TWEAK和Fn14的表達(dá)情況,了解TWEAK和Fn14與PDR的相關(guān)性。為確認(rèn)TWEAK表達(dá)與PDR臨床病理特征之間的關(guān)系,我們進(jìn)一步研究了視網(wǎng)膜細(xì)胞過(guò)表達(dá)TWEAK后,能夠提高該細(xì)胞增殖和膠原合成的能力。方法:玻璃體樣本的采集通過(guò)玻璃體手術(shù)分別獲得16個(gè)PDR病人和21個(gè)非PDR的2型糖尿病病人的玻璃體樣本。ELISA法檢測(cè)玻璃體液中TWEAK和Fn14含量通過(guò)ELISA法檢測(cè)兩組病人玻璃體液中TWEAK和Fn14的含量。Westernblot法分析玻璃體液中TWEAK和Fn14蛋白的表達(dá)量?jī)山M病人玻璃體液中TWEAK和Fn14蛋白的表達(dá)水平通過(guò)與GAPDH帶的灰度百分比表示。目的基因的獲取從293T細(xì)胞獲取人的總DNA,通過(guò)PCR技術(shù)獲得TWEAK擴(kuò)展片段。ARPE-19細(xì)胞培養(yǎng)和TWEAK過(guò)表達(dá)將ARPE-19細(xì)胞進(jìn)行貼壁培養(yǎng)。細(xì)胞融合大于90%時(shí),以1:3的比例分代。在ARPE-19細(xì)胞中過(guò)表達(dá)TWEAK。將TWEAK編碼序列克隆入pc DNA3.1(+)載體。將TWEAK-pc DNA3.1(+)和對(duì)照質(zhì)粒分別轉(zhuǎn)染入ARPE-19細(xì)胞。在G418液體中篩選并保存。RNA提取和定量RT-PCRRT-PCR操作按照試劑盒說(shuō)明書(shū)進(jìn)行,GAPDH作為內(nèi)對(duì)照。TWEAK和GAPDH引物由Sangon公司合成。Westernblot法分析細(xì)胞中TWEAK蛋白的表達(dá)量檢測(cè)過(guò)表達(dá)ARPE-19細(xì)胞在傳代不同階段,細(xì)胞中TWEAK蛋白的表達(dá)量。細(xì)胞計(jì)數(shù)檢測(cè),MTT檢測(cè)和群落形成檢測(cè)細(xì)胞計(jì)數(shù)檢測(cè)獲得細(xì)胞生長(zhǎng)曲線,MTT法檢測(cè)細(xì)胞活力,集落形成實(shí)驗(yàn)檢測(cè)細(xì)胞增殖能力。Westernblot法分析細(xì)胞中α-SMA,collagen I和collagen IV蛋白的表達(dá)量檢測(cè)過(guò)表達(dá)ARPE-19細(xì)胞在傳代不同階段,細(xì)胞中α-SMA,collagen I和collagen IV蛋白的表達(dá)量。統(tǒng)計(jì)和分析用Graph Pad Prism軟件進(jìn)行統(tǒng)計(jì)分析。數(shù)據(jù)結(jié)果表示為平均值加減標(biāo)準(zhǔn)差。兩組間對(duì)比采用t-test,多組均數(shù)對(duì)比采用單因素方差分析。P小于0.05視為有統(tǒng)計(jì)學(xué)意義。結(jié)果:PDR病人玻璃體液中TWEAK和Fn14水平升高21個(gè)非PDR的T2DM病人和16個(gè)PDR病人的玻璃體樣本被收錄本研究中。為確定TWEAK和Fn14與PDR的相關(guān)性,通過(guò)ELISA法檢測(cè)了兩組病人玻璃體液中TWEAK和Fn14的含量的變化,結(jié)果顯示,PDR病人玻璃體中TWEAK和Fn14含量顯著升高。通過(guò)Westernblot法分析了兩組病人玻璃體中TWEAK和Fn14的蛋白表達(dá)水平。結(jié)果顯示,TWEAK和Fn14在PDR病人玻璃體中的蛋白表達(dá)水平也隨之顯著升高,因此,我們認(rèn)為TWEAK/Fn14信號(hào)通路在PDR病人的玻璃體液中表達(dá)上調(diào)。穩(wěn)定的TWEAK過(guò)表達(dá)ARPE-19細(xì)胞系的建立從293T細(xì)胞中獲取人總DNA,利用PCR技術(shù)得到TWEAK編碼序列擴(kuò)展片段,然后被克隆入pc DNA3.1(+)載體,ARPE-19細(xì)胞被克隆的載體轉(zhuǎn)染,可以獲得穩(wěn)定表達(dá)TWEAK的ARPE-19細(xì)胞系。TWEAK過(guò)表達(dá)促進(jìn)了細(xì)胞增殖和膠原在ARPE-19中的合成為了研究TWEAK在ARPE-19細(xì)胞增殖中的作用,我們將ARPE-19(TWEAK過(guò)表達(dá))組和ARPE-19(陰性對(duì)照)組的細(xì)胞生長(zhǎng)曲線進(jìn)行對(duì)比,我們發(fā)現(xiàn),TWEAK過(guò)表達(dá)組細(xì)胞群在培育3天和5天時(shí)的生長(zhǎng)速度明顯加快。在培育24和48小時(shí)后,過(guò)表達(dá)組細(xì)胞的活力較對(duì)照組更強(qiáng)。無(wú)論初始培育的細(xì)胞數(shù)目是多少,TWEAK過(guò)表達(dá)組細(xì)胞形成的集落數(shù)目顯著多于對(duì)照組。因此我們認(rèn)為TWEAK過(guò)表達(dá)可以引起ARPE-19細(xì)胞的增殖和活力的增強(qiáng)。我們進(jìn)一步研究了纖維化相關(guān)分子在兩組細(xì)胞中的表達(dá)情況,結(jié)果顯示,過(guò)表達(dá)組細(xì)胞中α-SMA,collagen I和collagen IV的蛋白表達(dá)水平顯著高于對(duì)照組,并在細(xì)胞傳代過(guò)程中表達(dá)穩(wěn)定,提示TWEAK能提高纖維化相關(guān)分子在視網(wǎng)膜細(xì)胞中的表達(dá)。結(jié)論:在增殖性糖尿病視網(wǎng)膜病變病人的玻璃體中,TWEAK/Fn14通路表達(dá)上調(diào)。TWEAK/Fn14通過(guò)其相關(guān)機(jī)制推動(dòng)PDR的病理發(fā)展過(guò)程。TWEAK/Fn14通路在視網(wǎng)膜ARPE-19細(xì)胞增殖和膠原合成中的發(fā)揮調(diào)節(jié)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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.2;R774.1

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