整合素连接激酶对视网膜色素上皮细胞生长、凋亡和分泌功能的影响

Influence of integrin-linked kinase on the proliferation, apoptosis and secretory function of human retinalpigment epithelial cell in vitro

背景 玻璃体视网膜手术后视网膜色素上皮（RPE）细胞的增生和移行是增生性玻璃体视网膜病变（PVR）发生的主要病理机制,干预RPE细胞的生物学行为对于PVR的靶向治疗有重要意义. 目的 观察整合素连接激酶（ILK）对RPE细胞生长、凋亡及分泌血管内皮生长因子（VEGF）的影响. 方法 用低糖DMEM培养基常规培养和传代人RPE D407细胞系,选择50代以内的细胞进行实验.应用特异性敲减ILK的小干扰RNA（siRNA） （ILK-siRNA）与100μl无血清培养基和RPE细胞共培养的方式转染RPE细胞作为实验组,应用非特异性siRNA转染RPE细胞作为对照组,分别将24孔板置于37℃、体积分数5％ CO2孵箱中孵育.转染48 h后,应用MTT比色法检测各组RPE细胞活力；应用流式细胞仪检测RPE细胞的生长周期及细胞凋亡情况；采用Western blot法检测并比较各组RPE细胞中cyclin D1和caspase-3表达水平的变化；应用ELISA法检测并比较各组RPE细胞分泌VEGF水平的变化. 结果 转染siRNA 48 h后,MTT法检测见实验组和对照组RPE细胞活力分别为（43.69±0.89）％和（73.95±1.20）％,实验组比对照组降低了40.92％,差异有统计学意义（t=49.524,P=0.000）.流式细胞术检测发现,实验组处于G1期的细胞数为（83.30±1.26）％,对照组为（47.10±0.93）％；实验组S期细胞数量显著减少,为（12.63±0.92）％,对照组为（36.25±1.21）％；实验组G2期细胞数量显著减少,为（4.07±1.40）％,对照组为（16.65±1.53）％,两组间G1、S、G2期细胞比例的差异均有统计学意义（t=56.624、-38.130、-14.860,均P=0.000）.实验组的细胞凋亡率为（15.18±1.22）％,对照组为（2.20±0.15）％,二者间差异有统计学意义（t=25.742,P=0.000）.Western blot 检测结果示,实验组cyclin D1的表达量明显降低,而caspase-3的表达量则明显增高.ELISA法检测结果示,实验组RPE细胞的VEGF分泌量为（1314.49±147.23） ng/L,比对照组的（3251.27± 113.87） ng/L降低了59.6％,差异有统计学意义（t=-31.217,P=0.000）.结论 体外培养的RPE细胞敲减ILK后导致细胞增生活力下降,凋亡增加,且RPE细胞分泌VEGF的功能受到抑制,有望为PVR的靶向防治提供理论基础.

Background The induction of proliferative and migratory potential in retinal pigmenepithelial （RPE） cellfollowing vitreouand retinal surgery ithe majocause of proliferative vitreoretinopathy （PVR）.Managing the biological behaviouof RPE celliimportanfothe targeting treatmenof PVR.Objective Thistudy wato investigate the effecof integrin-linked kinase（ILK） on the proliferation and apoptosiof human RPE celland theisecretion of vasculaendothelial growth factor（VEGF）in vitro.MethodThe RPE D407 line wacultured and passaged in loweglucose DMEM,and the cellwere used up to thei50th generation in the study.ILK-small interfering RNA（siRNA） transfected into RPE cellin DMEM waused athe experimental group,while nonspecifisiRNtransfected into RPE cellwaused athe control group.The viability of the cellwaassayed by MT48 houraftetransfection.The cell cycle progression and apoptosiof the cellwere examined using flow cytometry.The expression of cyclin D1 and caspase-3 proteinin RPE cellwaevaluated by Western blot,and VEGF level in the cell supernatanwadetermined by ELISA.The resultwere compared between the experimental group and the control group using the independensample test.Resu1tFourty-eighhouraftetransfection of ILK-siRNA,the viability of the RPE cellwa（43.69 ±0.89）% in the experimental group and（73.95 ± 1.20）% in the control group,showing decline of 40.92% in the experimental group compared to the control group（t=49.524,P=0.000）.In the experimental group,the proportionof cellin G1,and G2 phase were （83.30 ± 1.26） %,（12.63 ± 0.92） % and （4.07±1.40） %,respectively,and those in the control group were（47.10±0.93） %,（36.25±1.21） % and（16.65± 1.53）%,with significandifferencebetween the two groups（=56.624,-38.130,-14.860,all aP =0.000）.The apoptotirate was（15.18±1.22） % in the experimental group,which wasignificantly lowethan thain the control group（（2.20±0.15）%）（t=25.742,P =0.000）.In addition,the expression of the cyclin D1 protein reduced and thaof caspase-3 protein elevated in the experimental group compared with the control group.VEGF contenin the cell supernatanwa（1314.49± 147.23）ng/L in the experimental group and thain the control group wa（3251.27 ± 113.87）ng/L,showing reduction of 59.6% in the experimental group（t=-31.217,P=0.000）.ConclusionKnockdown of ILK in RPE cellinhibitthe proliferation and promoteapoptosiin human RPE cellin vitro.decreased level of ILK also reducethe secretion of VEGF in human RPE cells.These resultsuggesthathe knockdown of ILK in RPE cellmay be novel approach to the prevention and treatmenof PVR.