人类肾上皮细胞氧化损伤模型的建立

An oxidative injury model of human kidney epithelial cells

目的:采用过氧化氢(H2O2)对人类肾脏近端小管上皮细胞系(HKC)进行氧化损伤,建立损伤的细胞模型。方法:通过检测细胞活力、培养基中的丙二醛(MDA)含量和细胞内超氧化物歧化酶(SOD)活性的变化研究细胞损伤的程度;利用激光共聚焦显微镜观察细胞损伤前后骨桥蛋白(OPN)表达水平的变化;采用扫描电子显微镜(SEM)观察细胞损伤前后形貌和膜表面微结构的变化。结果:用1 mmol/L H2O2作用HKC不同时间后,细胞活力和SOD活性逐渐下降,MDA释放量增加,OPN表达量显著增加并在作用时间为1 h时达到最大值;经损伤后的细胞变得干瘪收缩,且膜表面粗糙,鞭毛、突触大部分断裂脱落。结论:H2O2能够明显损伤HKC,细胞损伤后,其活力和膜表面超微结构发生变化,并在膜上表达能黏附草酸钙晶体的OPN。因此,可用1 mmol/L H2O2作用HKC 1 h建立损伤模型。

AIM： To establish an injured cell model using human kidney proximal tubule epithelial cell line（HKC） to mimic the oxidative injury by hydrogen peroxide（H2O2）.METHODS： The cell viability,the content of malondialdehyde（MDA） in the culture supernatant and the activity of intracellular superoxide dismutase（SOD） were detected to investigate the degree of cell injury.Osteopontin（OPN） expressed on the cell membrane surface were observed by laser confocal microscopy before and after cell injury.The changes of cellular morphology and the ultrastructure of membrane surface were observed under scanning electronic microscope.RESULTS： After HKC cells were treated with H2O2 at the concentration of 1 mmol/L for different time,the cell viability and the activity of SOD decreased and the content of MDA increased.The expression level of OPN significantly increased and reached to maximae at 1 h.The injured cells appeared shriveled and rough surface,and the shedding of most flagellae was also observed.CONCLUSION： H2O2 induces severer injury in HKC cells,including not only the cell viability and membrane surface ultrastructure,but also the OPN expression on the membrane,which could bind calcium oxalate crystal.Therefore,treatment with H2O2 at the concentration of 1 mmol/L for 1 h can be used to establish an oxidative injury model in HKC cells.