高渗应激对肝细胞癌Huh7细胞增殖和凋亡的影响

Effect of hyperosmotic stress on proliferation and apoptosis of hepatocellular carcinoma Huh7 cells

目的观察高渗应激对肝细胞癌Huh7细胞增殖和凋亡的影响，并探讨其调控机制。方法免疫组织化学法检测64例肝细胞癌组织中渗透压调控的转录因子活化T细胞核因子5（NFAT5）的表达；氯化钠模拟肿瘤高渗微环境，Westernblot法检测NFAT5蛋白的表达；流式细胞仪检测细胞凋亡；噻唑蓝（MTF）比色法检测细胞体外增殖活力。结果癌组织NFAT5的阳性表达率为9．38％，明显低于癌旁组织（68．75％，P〈0．01）。高渗24h组（100mmol／LNaCl）能使Huh7细胞NFAT5蛋白的表达显著升高（P〈0．01）。高渗24h组（100mmol／LNaCl）细胞凋亡率[（26．0±3．2）％]明显高于空白对照组[（0．8±0.2）％，P〈0．01]。高渗24h（100mmol／LNaCI）能明显抑制Huh7细胞增殖．细胞生长抑制率为（31．35±2．09）％（P〈0．01）。结论作为哺乳动物唯一的渗透压调节相关因子NFAT5，肝细胞癌组织与癌旁组织在NFAT5高表达率上差异有统计学意义，高渗应激能上调肝癌细胞NFAT5的表达，诱导肝癌细胞凋亡，并抑制细胞增殖。

Objective TO investigate the effect of hyperosmotic stress on the proliferation and ap- optosis of hepatocellular carcinoma Huh7 cells, and preliminarily explore the mechanism. Methods The expression of m.ammalian osmotic regulator nuclear factor of activated T-cells 5 （ NFAT5 ） was detected in 64 cases of hepAtocellular carcinoma by using immunohistochemistry. NaCI was used as a chemical hyper- osmotic-inducibie reagent to mimic tumor hyperosmotic microenvironment. The expression of NFAT5 pro- tein was analyzed by Western blotting. Cell apoptosis was examined by using flow cytometry and cell growth was measured by methyl-thiazol-tetrazolium （MTY） assay. Results NFAT5 positive expression rate was low （9. 38% ） in hepatocellular carcinoma tissue but high in the corresponding peritumoral tissue （68. 75% ） （P 〈0.01 ）. Under hyperosmotic stress （100 mmol/L NaC1） for 24 h, the protein expression levels of NFAT5 in Huh7 cells were up-regulated （P 〈 0. 01 ）. The apoptosis rate in experimental group [ （26.0±3.2 ） % ] was significantly higher than that in blank control group [ （ 0. 8±0. 2 ） % ]（ P 〈 0. 01 ）. Hyperosmotic stress （ 100 mmol/L NaC1） also inhibited growth of Huh7 cells with the cell growth inhibitory rate being （31.35±2.09） % （ P 〈 0. 01 ）. Conclusion There are obvious differences in the ex- pression of the only mammalian osmotic regulator NFAT5 between hepatocellular carcinoma tissue and per- itumoral tissue. Hyperosmotic stress can up-regulate the NFAT5 protein expression, induce apoptosis of hepatocellular carcinoma Huh7 cells, and inhibit cells proliferation.