整合素连接激酶对人视网膜色素上皮细胞增殖的影响

目的研究整合素连接激酶(integrin linked kinase,ILK)小分子干扰RNA(siRNA)对人视网膜色素上皮(human retinal pigment epithelium,hRPE)细胞增殖的影响。方法培养hRPE细胞,角蛋白鉴定。以ILK为靶基因设计合成特异性的siRNA干扰片段转染hRPE细胞。荧光显微镜下观察转染效率,选择转染效率最高的干扰片段进行细胞转染。RT-PCR检测siRNA对ILK基因表达的抑制作用,MTT法检测转染前后hRPE细胞增殖活性。结果 ILK-siRNA可以成功转染至hRPE细胞,转染24h后hRPE细胞中ILK mRNA的相对表达水平在正常对照组、单纯脂质体组、阴性对照组及转染组中分别为0.44±0.48、0.43±0.38、0.42±0.47、0.32±0.71,正常对照组、单纯脂质体组和阴性对照组中ILKmRNA表达差异无统计学意义(P>0.05),转染组ILKmRNA的表达较正常对照组明显降低,差异有显著统计学意义(F=21.78,P<0.01)。不同浓度ILK-siRNA转染组在不同时间点细胞增殖较正常培养组明显降低(P<0.01),50nmol·L-1浓度转染组对hRPE细胞的生长抑制最明显。结论 hRPE细胞表达ILK,siRNA抑制ILKmRNA的表达,在一定范围呈时间浓度依赖性抑制hRPE细胞增殖。     

siRNA抑制缺氧人视网膜色素上皮细胞ILK的表达及对HIF-1α的影响

目的:探讨干扰RNA(siRNA)沉默缺氧培养下人的视网膜色素上皮细胞(hRPE)中整合素连接激酶(ILK)的表达及其对缺氧诱导因子1α(HIF-1α)表达的影响。方法:CoCl2建立hRPE细胞的化学缺氧模型,Western blot和RT-PCR方法半定量检测不同缺氧时间(0,6,12,24,48h)hRPE细胞中ILK、HIF-1α蛋白及其mRNA的表达;阳离子脂质体转染ILK的干扰片段抑制缺氧24h hRPE细胞中ILK的表达,同上方法检测转染后缺氧24h hRPE细胞中ILK的表达及其对HIF-1α表达的影响。结果:ILK表达于正常及缺氧培养的hRPE细胞中。随着hRPE细胞缺氧时间的延长,在蛋白和mRNA水平ILK、HIF-1α都呈现逐渐增加的表达趋势。siRNA抑制ILK在缺氧24h hRPE细胞中的表达,同时显著抑制了HIF-1α的表达,较阴性对照组、单纯脂质体组有显著差异(P<0.01)。结论:ILK可以通过HIF途径应答RPE细胞的缺氧反应。     

缺氧及小干扰RNA转染对人RPE细胞增生及血小板源性生长因子-BB表达的影响

背景 增生性玻璃体视网膜病变（PVR）为视网膜表面发生无血管、纤维细胞性增生膜,其发生和发展的具体机制尚未完全阐明.人视网膜色素上皮（hRPE）及血小板源性生长因子（PDGF）在PVR发展中的作用是近几年研究的热点. 目的 探讨缺氧对体外培养hRPE细胞PDGF-BB表达及增生的影响. 方法 hRPE细胞在6孔板中培养,实验组用10、15、20、30、40 μmol/L CoCl2模拟体外培养hRPE细胞的缺氧环境,对照组用未加CoCl2的培养液培养hRPE细胞,采用逆转录PCR（RT-PCR）与ELISA法检测PDGF-BB mRNA和蛋白的表达,采用MTT法检测hRPE细胞增生率.依据转染siRNA的不同将细胞分为PDGF-BB siRNA1组、PDGF-BB siRNA2组、PDGF-BB siRNA3组（为针对PDGF-BB的3条不同的siRNA,其中有一条为有效siRNA）、β-actin siRNA组、无关siRNA组和只加Lip2000的空白对照组.转染4～6h后,除空白对照组外,其余各组加入对细胞PDGF-BB mRNA和蛋白及对hRPE细胞增生影响最明显的15 μmol/L CoCl2模拟细胞缺氧环境24 h,检测PDGF-BB mRNA和蛋白及hRPE细胞增生率. 结果 未加CoCl2的对照组未检测出PDGF-BBmRNA和蛋白的表达,不同浓度CoCl2培养hRPE细胞PDGF-BB mRNA和蛋白的表达量不同,差异均有统计学意义（F=43.737,P＜0.01;F=54.612,P＜0.05）,15μmol/L CoCl2组PDGF-BB的表达量最多,与其他各组比较差异均有统计学意义（P＜0.05）.MTT检测结果显示,不同浓度CoCl2处理后PDGF-BB蛋白表达及hRPE细胞增生率明显不同,差异均有统计学意义（F=95.379,P＜0.01;F=63.375,P＜0.05）,15 μmol/L CoCl2组hRPE细胞增生率明显高于其他浓度组,差异均有统计学意义（P＜0.05）,PDGF-BB蛋白表达量与hRPE细胞增生率呈线性正相关（r=0.994,P＜0.05）.各细胞转染组hRPE细胞中PDGF-BB mRNA及蛋白的表达整体比较,差异均有统计学意义（F=156.330、125.650,P＜0.01）,且PDGF-BB siRNA2组PDGF-BB mRNA较其他各组明显下降,差异均有统计学意义（P＜0.01）.MTT检测结果显示,各细胞转染组PDGF-BB蛋白的表达及hRPE细胞增生率明显不同,差异均有统计学意义（F=73.131、98.564,P＜0.01）,PDGF-BB siRNA2组PDGF-BB蛋白的表达及细胞增生率与其他各组比较明显下降,差异均有统计学意义（P＜0.05）,PDGF-BB蛋白表达与hRPE细胞增生率呈线性正相关（r=0.996,P＜0.05）.结论 缺氧能够促进PDGF-BB的表达,PDGF-BB的表达上调可显著促进hRPE细胞的增生.在转染靶向PDGF-BB siRNA后,PDGF-BB的表达受到抑制,可有效降低hRPE细胞的增生率.     

抗PDGFR-α抗体对铁锈诱导的人视网膜色素上皮细胞增殖过程的作用

目的：观察抗血小板源性生长因子受体α（PDGFR-α）抗体对铁锈诱导的人视网膜色素上皮细胞（hRPE）增殖活性的影响。方法：采用细胞体外培养技术,用铁锈建立hRPE细胞增殖分化模型。设置空白组、铁锈组、抗PDGFR-α抗体（1,10,50,100μg/mL）处理组,分别作用0,12,24,48h后采用MTT比色法测定hRPE细胞的抑制率。结果：加入抗PDGFR-α抗体后hRPE细胞增殖活性在一定浓度范围内随着抗体浓度增加而下降,而50μg/mL抗PDGFR-α抗体为抑制hRPE细胞增殖的最佳浓度,其抑制率为42.44%。结论：抗PDGFR-α抗体可以抑制hRPE细胞的增殖活性。     

MicroRNA-96对人视网膜色素上皮细胞增殖与迁移的影响

目的：探讨MicroRNA-96（miR-96）对人视网膜色素上皮（RPE）细胞增殖和迁移的影响。方法：实验研究。通过RNA原位杂交检测人胚胎眼（20周）石蜡切片中RPE层miR-96的表达情况。将miR-96和阴性对照（NC）通过阳离子脂质体介导转染人眼RPE细胞,采用细胞增殖实验（MTS）、流式细胞术和Transwell实验分别检测细胞增殖、细胞周期以及细胞迁移能力。通过生物信息学及Western blot法确定miR-96作用的靶基因。应用Western blot检测miR-96对细胞增殖迁移相关信号通路蛋白（Akt、ERK）及细胞周期相关蛋白（p-Cdc2、CyclinD2、p-Rb）表达的影响。组间数据比较采用独立样本t检 验。结果：MiR-96在人眼RPE细胞中有表达。MTS结果显示,转染NC、miR-96后,人眼RPE细胞的相对增殖速率分别为100%、74%±2%,差异有统计学意义（t=42.174,P=0.002）。流式细胞术检测结果显示,转染miR-96后阻滞在G1期的RPE细胞显著多于转染NC后,且差异有统计学意义（t= -18.444,P=0.003）。Transwell实验结果显示,与转染NC相比,转染miR-96能显著抑制RPE细胞的迁移,差异具有统计学意义（t=6.754,P=0.002）。进而,明确了MITF是miR-96作用的靶基因。Western blot检测结果显示,转染miR-96后细胞中细胞周期相关蛋白p-Rb（t=11.211,P=0.002）、p-Cdc2（t=9.133, P=0.003）、CyclinD2（t=7.542,P=0.005）以及迁移相关信号通路蛋白p-ERK（t=16.699,P<0.001）,p-Akt （t=23.552,P<0.001）的表达水平均降低。结论：miR-96通过作用于靶基因MITF,并调控细胞周期和迁移相关蛋白的表达从而抑制人RPE细胞的增殖和迁移。     

RNA干扰抑制水通道蛋白1对血管内皮细胞迁移影响的研究

目的 探讨RNA干扰抑制水通道蛋白1(AQP-1)对血管内皮细胞迁移的影响以及角膜新生血管的形成机制.方法 实验研究.体外培养人血管内皮细胞,设计并合成特异性针对人AQP-1的小RNA干扰片段,用脂质体转染入血管内皮细胞,逆转录聚合酶链反应(RT-PCR)法检测RNA干扰效果,Transwell实验和损伤愈合实验观察血管内皮细胞迁移能力的改变.AQP-1 mRMA表达的组间比较采用单因素方差分析.Transwell细胞迁移实验和损伤愈合实验的结果采用独立样本的t检验.结果 转染24、48及72 h后AQP-1 mRNA表达下降,分别为正常对照组的11.3%、17.4%及29.7%,与正常对照组相比差异有统计学意义(P值分别为0.004、0.007、0.002).Transwell实验和损伤愈合实验提示转染AQP-1 siRNA后血管内皮细胞的迁移能力有明显下降,与正常对照组相比差异有统计学意义(t=10.813,P=0.016和t=22.431,P=0.027).结论 AQP-1特异性siRNA能有效抑制AQP-1的表达并明显降低血管内皮细胞的迁移能力,从而抑制角膜新生血管的形成.(中华眼科杂志,2008,44:741-744)     

HTRA1基因过表达对视网膜色素上皮细胞增殖及迁移影响

目的 研究HTRA1基因过表达对人视网膜色素上皮细胞(RPE)的增殖和迁移能力的影响,探讨HTRA1基因在老年黄斑变性(AMD)发病中的作用.方法 用携带人HTRA1基因的慢病毒感染体外培养的人RPE细胞,荧光显微镜下观察细胞的感染效率,RT-PCR和Westem Blot检测HTRAlmRNA和蛋白的表达,MTT比色法检测感染细胞毒性及细胞增殖能力,TransweH小室检测细胞迁移能力变化.结果 与空载体慢病毒感染RPE细胞相比,HTRA1基因慢病毒感染的RPE细胞HTRA1mRNA和蛋白的表达量明显增加,细胞增殖能力及迁移能力均明显下降,差异有统计学意义(P＜0.01).结论 过表达HTRA1基因能抑制人视网膜色素上皮细胞的增殖及迁移,THRA1基因可能通过影响RPE细胞功能参与AMD的进程.     

ILK siRNA对高浓度葡萄糖诱导的人LECs增生和上皮向间质转化的抑制作用

目的探讨整合素连接激酶（ILK）对高浓度葡萄糖诱导的人晶状体上皮细胞（LECs）增生和转化的作用。方法将体外培养的人LECs系SRA01/04细胞分别培养在含葡萄糖5.5mmol/L（正常对照组）和30.5mmol/L（高糖组）的培养液中,用RT-PCR检测培养0、6、24h后ILK mRNA的表达;用ILK siRNA脂质体转染细胞,转染6h后,加入2组培养液处理24h,检测ILK、细胞增生核抗原（PCNA）、LECs转化指标α-SMA和FN在mRNA水平的表达。结果高糖组培养6h和24h,SRA01/04细胞ILK mRNA是正常对照组的2.48倍和2.32倍（P〈0.01）,刺激后24h,SRA01/04细胞PCNA、α-SMA、FN的mRNA表达分别是正常对照组的1.75、1.96和1.75倍（P〈0.01）。ILK siRNA干扰后,正常对照组ILK的表达是非转染细胞的30%,高糖组转染细胞ILK mRNA水平（P〈0.01）是非转染细胞的21%（P〈0.01）,转染细胞PCNA、α-SMA和FN的表达分别是非转染细胞的29%、33%和39%（P〈0.01）。结论高浓度葡萄糖可诱导LECs增生、上皮向间质转化及ILK表达上调,抑制ILK的高表达能阻止这些过程。     

shRNA抑制缺氧条件下人视网膜色素上皮细胞低氧诱导因子-1α基因对血管内皮生长因子表达的影响

目的探讨缺氧条件下人视网膜色素上皮（hRPE）细胞低氧诱导因子-1α（HIF-1α）对血管内皮生长因子（VEGF）表达的影响。方法利用体外转录法合成针对HIF—1α mRNA序列的靶点之一的小发卡环RNA（shRNA）,以化学低氧诱导剂CoCl2模拟RPE细胞缺氧环境,对缺氧培养条件下hRPE细胞的HIF-1α进行RNA干扰,通过逆转录聚合酶链反应（RT-PCR）检测HIF-1α和VEGF mRNA的表达,免疫印迹法检测HIF—1α和VEGF蛋白水平,以正常组、缺氧组及阴性转染组作为对照,观察其对HIF-1α基因的沉默效果及对VEGF表达的抑制作用。结果转染HIF-1α mRNA的特异性shRNA后,RT-PCR检测结果显示缺氧条件下hRPE细胞HIF-1α基因沉默效果为77．1％,VEGF mRNA的表达水平下降了27．8％;免疫印迹法检测结果显示HIF—1α和VEGF蛋白水平显著降低。结论针对HIF-1α mRNA的shRNA能有效地使HIF-1α基因沉默,进而抑制缺氧对VEGF的上调作用。（中华眼科杂志．2007。43：1022—1027）     

基质细胞衍生因子-1α对体外培养人视网膜色素上皮细胞增殖的影响研究

目的:研究基质细胞衍生因子-1α(stromal cell-derived fac-tor-1α,SDF-1α)对体外培养的人类视网膜色素上皮(hu-man retinal pigment epithelium,hRPE)细胞增殖的影响。方法:应用MTT法研究SDF-1α对体外培养的人RPE细胞增殖的影响;用细胞化学染色法检测添加SDF-1α前后人视网膜色素上皮细胞内细胞增殖核抗原(proliferatingcell nucler antigen,PCNA)的表达情况。结果:MTT法表明SDF-1α可促进体外培养的hRPE细胞的增殖(P<0.05);细胞化学电镜下观察发现:添加SDF-1α前hRPE细胞未表达或低表达PCNA,而添加SDF-1α后细胞高度表达PCNA。结论:细胞因子SDF-1α可促进体外培养的hRPE细胞增殖。     

HSP47 siRNA对体外培养HTCF细胞生物学行为及TGF-β1表达水平的影响

目的:探究热休克蛋白47(HSP47)siRNA对体外培养人眼Tenon囊成纤维(HTCF)细胞生物学行为及转化生长因子-β1(TGF-β1)表达水平影响。方法:体外培养HTCF细胞,并分为:空白对照组、空载体组和转染组;转染组根据HSP47基因序列设计并合成干扰siRNA序列,构建载体并导入HTCF细胞中;空载体组导入空白载体。采用RT-PCR和蛋白质印迹实验检测细胞中HSP47 mRNA和蛋白的表达情况,采用克隆形成实验、流式细胞术、Transwell法及划痕实验检测细胞增殖、凋亡、侵袭及迁移,蛋白质印迹实验检测增殖、凋亡、侵袭、迁移蛋白和TGF-β1的表达情况。结果:相比空载体组,转染组HSP47 mRNA和蛋白的表达、克隆形成率、细胞愈合率、侵袭细胞数目、Ki67、N-cadherin、TGF-β1蛋白相对表达水平显著降低(P<0.05),E-cadherin蛋白相对表达水平显著升高(P<0.05),但细胞凋亡率、Bcl-2和Bax蛋白相对表达水平均无差异(P>0.05)。结论:HSP47 siRNA可以通过抑制TGF-β1蛋白的表达降低HTCF细胞的增殖、侵袭及迁移能力,但对HTCF细胞的凋亡无明显影响。     

Targeting of integrin-linked kinase with a small interfering RNA suppresses progression of experimental proliferative vitreoretinopathy

Integrin-linked kinase (ILK) is a serine/threonine kinase that interacts through its COOH terminus with β1 and β3 integrins, which mediates a diversity of cell functions by coupling integrins and growth factors to cascades of downstream signaling events. The purpose of this work was to investigate the effects of ILK on development of experimental proliferative vitreoretinopathy (PVR). Cultured human RPE cell line D407 was knocked down for ILK using a small interfering RNA (siRNA). For this, cellular ILK expression was quantified by real-time quantitative PCR, Western blot analysis and immunocytochemical assay, and cytotoxicity of transfection was determined by MTT assay. Moreover, cell attachment, spreading, migration, microfilament dynamics, and cell cycling assays were performed. Furthermore, the impact of the ILK-specific siRNA on PVR was tested using a rabbit model in which PVR was induced by the injection of human RPE cells. Prevalence of PVR and retinal detachment were determined by indirect ophthalmoscopy on days 1, 3, 7, 14, 21 and 28 post-injection. The results showed that blocking the expression of ILK by siRNA significantly inhibited human RPE cell attachment, spreading, migration and proliferation. The knockdown of ILK also disturbed F-actin assembly and induced a cellular arrest in the G1 phase of the cell cycle. Though the eyes injected with ILK-specific siRNA also developed features of PVR, the severities of day 28 post-injection were significantly lower than those in the control eyes (P < 0.01). We conclude that targeting of ILK with a small interfering RNA not only inhibits human RPE cell attachment, spreading, migration and proliferation in vitro, but also effectively suppresses development of proliferative vitreoretinopathy in a rabbit model. This may be a potential therapeutic usefulness in treating PVR.     

Targeting of integrin-linked kinase with a small interfering RNA suppresses progression of experimental proliferative vitreoretinopathy

Integrin-linked kinase (ILK) is a serine/threonine kinase that interacts through its COOH terminus with β1 and β3 integrins, which mediates a diversity of cell functions by coupling integrins and growth factors to cascades of downstream signaling events. The purpose of this work was to investigate the effects of ILK on development of experimental proliferative vitreoretinopathy (PVR). Cultured human RPE cell line D407 was knocked down for ILK using a small interfering RNA (siRNA). For this, cellular ILK expression was quantified by real-time quantitative PCR, Western blot analysis and immunocytochemical assay, and cytotoxicity of transfection was determined by MTT assay. Moreover, cell attachment, spreading, migration, microfilament dynamics, and cell cycling assays were performed. Furthermore, the impact of the ILK-specific siRNA on PVR was tested using a rabbit model in which PVR was induced by the injection of human RPE cells. Prevalence of PVR and retinal detachment were determined by indirect ophthalmoscopy on days 1, 3, 7, 14, 21 and 28 post-injection. The results showed that blocking the expression of ILK by siRNA significantly inhibited human RPE cell attachment, spreading, migration and proliferation. The knockdown of ILK also disturbed F-actin assembly and induced a cellular arrest in the G1 phase of the cell cycle. Though the eyes injected with ILK-specific siRNA also developed features of PVR, the severities of day 28 post-injection were significantly lower than those in the control eyes (P < 0.01). We conclude that targeting of ILK with a small interfering RNA not only inhibits human RPE cell attachment, spreading, migration and proliferation in vitro, but also effectively suppresses development of proliferative vitreoretinopathy in a rabbit model. This may be a potential therapeutic usefulness in treating PVR.     

TRAF6基因沉默对人视网膜母细胞瘤细胞增殖、凋亡和侵袭的影响

目的：探讨体外沉默肿瘤坏死因子受体相关因子6(TRAF6)基因对人视网膜母细胞瘤Y79细胞增殖、凋亡和侵袭的影响。

方法：靶向TRAF6基因的小干扰RNA(TRAF6 siRNA)转染Y79细胞,采用qRT-PCR和Western blot法检测转染效果,MTT比色法及细胞克隆实验测定沉默TRAF6对Y79细胞增殖的影响,流式细胞术检测细胞凋亡及细胞周期,Transwell小室检测细胞迁移和侵袭能力。

结果：Y79细胞经TRAF6沉默后,TRAF6 mRNA和蛋白表达水平与对照组相比均明显降低,细胞存活率、克隆形成率均明显低于对照组细胞,细胞凋亡率明显高于对照组,同时细胞周期发生明显变化,G0/G1期细胞数目增多,S和G2/M期细胞数目减少,且侵袭细胞数目、迁移细胞数目相比对照组明显减少。

结论：沉默TRAF6后能显著抑制视网膜母细胞瘤Y79细胞的生长,促进肿瘤细胞的凋亡,同时抑制其侵袭和迁移能力,TRAF6可能是视网膜母细胞瘤治疗的新靶点。     

Inhibition of cell proliferation, migration and apoptosis in blue-light illuminated human retinal pigment epithelium cells by down-regulation of HtrA1

AIM: To investigate the effect of HtrA1 on the proliferation, migration and apoptosis of human retinal pigment epithelium (RPE) cells in the light injured model, as well as the expression of the apoptosis related molecules. METHODS: The human RPE cell line ARPE-19 was exposed to blue light to establish the light injured model. The cells were transfected with HtrA1 siRNA to knockdown HtrA1 expression. Subsequent expression of HtrA1 was determined by real-time polymerase chain reaction (RT-PCR) and Western blot, respectively. Changes in cell proliferation, migration and apoptosis were assessed by cell counting kit-8 (CCK-8), Transwell assay and flow cytometry respectively, as well as changes in the mRNA and protein levels of Bax, Caspase-3 and Bcl-2 expression. RESULTS: HtrA1 was highly expressed in ARPE-19 cells after blue light irradiation. Knockdown of HtrA1 expression inhibited the proliferation, migration and apoptosis of the blue-light-irradiated ARPE-19 cells (P<0.05). Bax and Caspase-3 expression were significantly reduced both at mRNA and protein levels (P<0.05) after siRNA treatment. Bcl-2 expression significantly increased in blue-light-irradiated ARPE-19 cells after siRNA interference (P<0.05). CONCLUSION: Silence of HtrA1 may inhibit the proliferation, migration and apoptosis of ARPE-19 cells in light injured model. Moreover, HtrA1 suppression in blue-light-irradiated ARPE-19 cells may ameliorate cell apoptosis through down-regulation of Bax and Caspase-3, and up-regulation of Bcl-2 expression.