雷帕霉素对转化生长因子-β2诱导的人晶状体上皮细胞上皮-间叶样转化的抑制作用及其机制  

Effects of rapamycin on transforming growth factor-β2-induced epithelial-myofibroblast transition of human lens epithelial cells

作　　者：林婷婷[1] 王思敏[1] 刘加勇[1] 冯浩[1] 宁宏[1] 

机构地区：[1]中国医科大学附属第一医院眼科,沈阳110001

出　　处：《中华实验眼科杂志》2013年第4期347-351,共5页Chinese Journal Of Experimental Ophthalmology

基　　金：辽宁省科技计划项目（2008225016）

摘　　要：背景转化生长因子-β2（TGF-β2）诱导的人晶状体上皮细胞（LECs）上皮细胞-肌成纤维细胞转化（EMT）过程是后发性白内障（PCO）的主要发病机制，因此寻求有效抑制这一作用的药物对于防治PCO有重要意义。目的观察雷帕霉素（RAPA）对人LECs增生和TGF-β2诱导的EMT的抑制作用。方法采用含质量分数10％胎牛血清的DMEM高糖细胞培养液对人LECs系SRA01／04进行体外培养，换以无血清培养液培养后分别用TGF-β2（5mg／L）、TGF-β2＋10nlg／LRAPA、TGF-β2＋100mg／LRAPA、TGF-β2＋1000mg／LRAPA、TGF-β2＋10000mg／LRAPA共培养SRA01／0472h，仅用无血清培养液培养的SRA01／04作为对照组。采用MTT法检测各组SRAOI／04的吸光度（A490）值以评估SRA01／04的增生情况，并计算RAPA对细胞生长的抑制率。各组细胞作用48h后，采用逆转录PCR（RT-PCR）法和Westernblot法分别检测LECsETM的标志物-α平滑肌肌动蛋白（d-SMA）和上皮性钙黏附蛋白（E-cad）的表达。选取TGF-β2＋400mg／LRAPA分别作用于培养的SRA01／0424、48、72h，采用Westernblot法检测SRA01／04中α-SMA、E-cad的表达水平。结果MTT法检测结果显示，对照组、TGF-β2组、TGF-β2＋10ing／LRAPA组、TGF-β2＋100mg／LRAPA组、TGF-β2＋1000ing／LRAPA组和TGF-β2＋10000mg／LRAPA组SRA01／04的A490值分别为0．680＋0．020、0．550±0．013、0．480＋0．014、0．400＋0．011和0．200＋0．019，表明随着RAPA质量浓度的增加，SRAOI／04增生值降低，差异有统计学意义（F=101．920，P=0．000），且RAPA的抑制率逐渐增加。RT-PCR和Westernblot检测结果表明，阴性对照组SRAOI／04中α-SMAmRNA（α-SMAmRNA／β-actinmRNA）及其蛋白（α-SMA／β-actin）仅有少量表达，TGF．B，组可见SRAOI／04中01．SMAmRNA及其蛋白表达量明显增加，但不同质量浓度的RAPA作用于SRA01／04后，随着RAPA质量浓度的增加，SRAOI／04中α-SMAmRNA及蛋白的表达�Background Epithelial-myofibroblast transition （EMT） of human lens epithelial ceils （LECs） induced by transforming growth factor-J32 （TGF-[32 ）is the main mechanism in the pathogenesis of posterior capsular opacification（PCO）. Seeking an effective drug capable of inhibiting this process is important for the prevention and treatment of PCO. Objective The purpose of this study was to investigate the inhibitory effect of rapamycin （RAPA）on the proliferation of human LECs and TGF-132-induced EMT. Methods Human LEC strain（SRA01/04） was cultured in DMEM with high glucose and 10% fetal bovine serum. The cells were consequently cultured in serum- free DMEM with 5 mg/L TGF-β2 ,TGF-β2 ＋ 10 mg/L RAPA, TGF-β2 ＋ 100 mg/L RAPA, TGF-β2 ＋ 1000 mg/L RAPA or TGF-β2 ＋10 000 mg/L RAPA for 72 hours, and SRA01/04 cultured in serum-free DMEM were used as control. The proliferation rate（A490 ） of SRA01/04 in the different groups was detected using the MTT assay and the rate of inhibition of RAPA was calculated. The expressions of the α-smooth muscle actin（α-SMA） and E-cadherin （E-cad）mRNA and protein were detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot, respectively. The changes in the expression of α-SMA and E-cad in SRA01/04 were evaluated by Western blot 24,48 and 72 hours after TGF-132 ＋400 mg/L RAPA treatment. Results The A490 value of SRA01/04 was 0. 680±0. 020, 0. 550±0. 013,0. 480±0. 014,0. 400±0. 011 and 0. 200±0. 019 in the control group,TGF-β2 group,TGF-β2 ＋ 10 mg/L RAPA group,TGF-β2 ＋ 100 mg/L RAPA group,TGF-β2 ＋ 1000 mg/L RAPA and TGF-β2＋ 10 000 mg/L RAPA group, respectively, showing a gradually declining trend in SRA01/04 rate of proliferation with increasing RAPA concentrations （ F = 101. 920, P = 0. 000）. RT-PCR and Western blot assay showed that the relative expression levels of α-SMA mRNA（ α-SMA mRNA/β-actin mRNA）and protein（α-SMA/β-actin） in the cells were significantly increased in the TG

关 键 词：雷帕霉素 转化生长因子-Β2 人晶状体上皮细胞 上皮细胞一肌成纤维细胞转化 

分 类 号：R776.1[医药卫生—眼科]