HES1在小梁网细胞中作用的初步研究

Preliminary study on the role of HES1 in human trabecular meshwork cells

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 sh RNA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p LKO.1-puro sh RNA慢病毒载体构建HES1 sh RNA质粒。Lipofectamine 2000慢病毒包装法感染原代HTMCs,使其稳定表达HES1sh RNA/Scramble质粒。采用q-PCR和Western blot方法检测HES1sh RNA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 sh RNA有效下调HES1的m RNA和蛋白水平表达(P<0.05)。同时,HES1 sh RNA组促纤维化ECM(FN;COL1;α-SMA)的m RNA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 sh RNA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 sh RNA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 HEK293PT were cultured and passaged in vitro, and infected with HESlshRNA/Scramble lentivirns. 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.