目的:利用慢病毒包装系统使人眼小梁网细胞(HTMCs)稳定表达HES1 shRNA并初步探索HES1在HTMCs的作用.方法:体外培养HTMCs和HEK293FT细胞并进行传代,同时应用pLKO.1-puro shRNA慢病毒载体构建HES1 shRNA质粒.Lipofectamine 2000慢病毒包装法感染原代HTMCs,使其稳定表达HES1 shRNA/Scramble质粒.采用q-PCR和Western blot方法检测HES1shRNA的敲低效果及促纤维化细胞外基质蛋白(ECM)的表达变化;HTMCs增殖和迁移能力分别通过CCK-8细胞活性分析和Transwell计数实验进行分析.结果:原代细胞经过培养后贴壁生长,呈长梭形,生长状态良好,形态符合HTMCs形态学特征.HES1 shRNA有效下调HES1的mRNA和蛋白水平表达(P＜0.05).同时,HES1 shRNA组促纤维化ECM(FN;COL1;α-SMA)的mRNA和蛋白表达水平较Scramble组显著降低(P＜0.05).而Transwell计数实验显示HES1 shRNA组HTMCs的迁移数量较Scramble组增加(P＜0.05);CCK-8细胞活性分析实验表明抑制HES1表达对HTMCs的增殖活性影响不大,差异不具有统计学意义(P＞0.05).结论:成功建立了稳定表达HES1 shRNA的HTMCs细胞株.HES1可以影响HTMCs中促纤维ECM的生成及HTMCs的增殖和迁移能力.%Objective:To establishhuman trabecular meshwork cells (HTMCs) cell line with stable HES1 shRNA expression and explore the role of Hairy and enhancer of split-1 (HES1) in HTMCs.Methods:Primary HTMCs and HEK293FT were cultured and passaged in vitro,and infected with HES1shRNA/Scramble lentivirus.The effects of HES1 knockdown on HES1 and profibrotic extracelluar matrix (ECM) expression were analyzed through q-PCR and Western blot.Transwell and CCK-8 counting assay were used to examine the ability of migration and proliferation HTMCs.Results:The cultured primary HTMCs were in a fibrocyte-like form with long fusiform shape and grew well.HTMCs infected with HES1 shRNA exhibited a significant decrease in HES1 and profibrotic ECM (fibronectin,FN;collagen I,COL1;alpha-smooth muscle actin,α-SMA) expression.HES1 shRNA treatment also increased HTMCs migration and proliferation.Conclusion:HTMCs with stable HES1 shRNA expression can be successfully built.And HES1 could affect ECM expression and celluar functions in HTMCs.
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