叔丁基过氧化氢诱导小梁网细胞氧化应激模型的建立

In vitro Study on t BHP Oxidative Stress Model of Human Trabecular Meshwork Cells

目的体外建立叔丁基过氧化氢小梁网细胞氧化应激模型。方法应用t BHP刺激体外培养的小梁网细胞(i HTM)后,观察小梁网细胞形态学的改变,分别用MTT法检测氧化损伤后小梁细胞的活性,流式细胞术检测活性氧水平,SucLLVY-荧光底物检测糜蛋白酶样蛋白酶体的活性,流式细胞术和激光共聚焦显微镜检测小梁细胞的凋亡。结果正常培养的小梁细胞呈梭形,折光性好,贴壁较牢;应用t BHP刺激后,部分细胞变圆、脱落、皱缩,小梁细胞的细胞活性明显下降,活性氧水平升高,糜蛋白酶样蛋白酶体活性降低,细胞凋亡水平明显增加。随着刺激时间的延长,上述改变更为明显。结论 t BHP刺激后,小梁网细胞出现典型的氧化应激改变,t BHP刺激小梁网模型的建立可应用于青光眼氧化应激的研究。

Objective To establish the oxidative stress model of immortalized human trabecular meshwork cells （iHTM） induced by tert -butyl hydroperoxide （tBHP）. Methods iHTM were cultured and treated with tBHP （2001xmol/L） for 1 and 2h. The 3 - （4,5 - dimethyl - thiozol - 2 - yl） - 2,5 - diphenyl - tetrazo - tetrazolium bromide （MTT） assay was used to analyze the cell viability. The 2＇, 7＇ - Dichlorofluo - rescin - diacetate （ DCFH - DA） was used to determine the reactive oxygen species （ROS） levels, and the succinyl - leucine - leucine - valine - tyrosine - aminoluciferin （ Suc - LLVY） substrate was used to measure the chymotrypsin - like protease activi- ties. Cell apoptosis was analyzed by Hoechst33258 staining and Annexin V -fluorescein isothiocyanate （FITC） /propidium iodide （PI） labeling. Results After tBHP treatment, the iHTM cell viability decreased obviously, while the ROS levels, chymotrypsin proteasome activity, and the cell apoptosis increased significantly. Longer duration of tBHP, the changes were more apparent. Conclusion The iHTM displayed typical oxidative changes after stress by tBHP. The iHTM oxidative stress model induced by tBI-IP was suitable for study glaucoma.