PEDF对人晶状体上皮细胞生长调控的体内外研究

Effects of PEDF on human lens epithelial cells growth regulation in vivo and in vitro

目的:探讨在体内及体外色素上皮衍生因子(PEDF)对人晶状体上皮细胞(LEC)生长的调节作用。方法:体内研究用ELISA法检测年龄相关性白内障患眼房水PEDF浓度,术中取前囊膜标本检测LEC密度,分析PEDF水平与LEC数量、白内障类型的关系;体外研究,加PEDF培养人晶状体上皮细胞株(HLE-B3),CCK8法检测细胞生长活性,流式细胞仪通过Annexin VFITC/7-AAD双染法检测细胞周期和凋亡变化。结果:120例白内障患眼房水PEDF浓度为(156.2±41.2)ng/ml,中央区前囊下LEC密度随房水PEDF水平下降而降低(r=0.556,P<0.01),控制年龄因素后仍呈正相关(r=0.387,P<0.05),皮质型白内障房水PEDF浓度较核硬化型更低(P<0.05);体外培养的LEC细胞株加入PEDF后,细胞生长明显加快(F=187.33,P<0.01),细胞凋亡被显著抑制,凋亡率从13.50%降为2.08%(t=7.90,P<0.01),细胞分裂活性增强,G2期+S期细胞比例从28.54%升为54.05%(t=6.32,P<0.01)。结论:人眼内PEDF可能作为LEC营养因子发挥细胞周期调控和抗凋亡的重要作用,参与晶状体生理性状维持和白内障发生发展。

Objective：To investigate in vivo and in vitro the role of PEDF on human lens epithelial cells biological behaviour regulation. Methods：In vivo study,aqueous PEDF concentration was detected by enzyme-linked immunosorbent assay （ELISA） in age-related cataract patients who underwent cataract phacoemulsification extraction surgery. The relationship of PEDF level was analyzed with LECs density on anterior lens capsular specimens and cataract types. In vitro study,PEDF was added in culture media of human lens epithelial cell line （HLE-B3）. The growth activity of HLE-B3 was detected by Cell Counting Kit （CCK8）. Flow cytometry with AnnexinV-FITC/7-AAD double staining method was used to detect the changes of cell cycle and apoptosis. Results： The mean aqueous PEDF concentration of 120 cataract cases was （156.2 ± 41.2）ng/ml. The central anterior subcapsular LEC density significantly decreased with aqueous PEDF level reducing（r = 0.556,P 〈 0.01 ）, and the positive correlation remained after controlling the age factor （r = 0.387,P 〈 0.05）. PEDF concentration in aqueous humor of cortical cataract was lower than that of nuclear sclerosis （P 〈 0.05）. After adding PEDF in culture medium of HLE-B3 line, cell＇s growth accelerated significantly （F = 187.33,P 〈 0.01）,and apoptosis was significantly inhibited （the apoptosis rate fell from 13.50% to 2.08% ,t = 7.90,P 〈 0.01）,and the proliferative division activity increased （cell ratio in G2＋S phase rose from 28.54% to 54.05% ,t = 6.32,P 〈 0.01）. Conclusion：In human eyes,PEDF may function as LEC cytotrophic factor to play the important biological effects of cell cycle regulation and anti- apoptosis,involved in maintenance of ienticular physiological state and cataract development.