华蟾毒精抑制人肝癌细胞株增殖、促凋亡作用及机制探讨

Effect of cinobufagin on Proliferation Inhibition and Apoptosis of Human Hepatocellular Carcinoma Cell Line

目的:观察华蟾毒精对人肝癌细胞株增殖抑制及促凋亡作用,并探讨其机制。方法:选取人肝癌细胞HepG2进行培养,取对数生长期HepG2细胞分为实验组1(浓度为25μmol/L华蟾毒精处理)、实验组2(浓度为50μmol/L华蟾毒精处理)、实验组3(浓度为100μmol/L华蟾毒精处理)和对照组(加等体积的生理盐水),培养24h、48h后采用MTT法和流式细胞术检测对照组和实验组肝癌细胞HepG2增殖抑制率和凋亡率。培养48h后荧光定量PCR检测AKT、mTOR、Caspase3、Bax和Bcl-2 m RNA表达量,免疫印迹法检测Caspase3、Bax和Bcl-2蛋白表达量。结果:培养24h、48h,实验组1、实验组2和实验组3细胞增殖抑制率均显著高于对照组(P<0.05),且实验组间两两比较差异均有统计学意义(P<0.05);培养24h、48h实验组1、实验组2和实验组3细胞凋亡率均显著高于对照组(P<0.05),实验组间两两比较差异均有统计学意义(P<0.05);培养48h,实验组1、实验组2和实验组3细胞AKT、mTOR、Bcl-2 m RNA表达量与对照组相比均显著降低(P<0.05),Bax和Caspase-3 m RNA表达量均显著增加(P<0.05),且实验组Caspase3、Bax和Bcl-2蛋白表达与m RNA表达结果趋势一致。结论:华蟾毒精能够抑制肝癌细胞HepG2增殖,并促进其凋亡,作用机制可能与通过下调Bcl-2蛋白和上调Bax、Caspase-3蛋白的表达,调控AKT/mTOR信号通路有关。

Objective ：To observe the effect and mechanisnl of cinobufagin on proliferation and pro - apoptotic of human hepatoma cell line. Methods ： Human hepatoma cell line HepG2 was cultured, and the HepG2 cells were divided into the experimental group 1 （ added 25 μmol/L cinobufagin）, the experimental group 2 （added 50 μmol/L cinobufagin）, the experimental group 3 （added 100 μmol/L cinobufagin） and the control group （ added equal volume of saline）. The proliferation inhibition rates were measured by MTT assay and the apoptotic rates were determined by flow cytometry in the control group and the experimental group after 24h and 48h of culture. The mRNA expressions of AKT, roTOR, Caspase3, Bax and Bcl - 2 were detected by Real time Quantitative PCR and the protein expressions of Caspase3, Ban and Bcl - 2 were detected by Western blotting. Results After 24h and 48h of culture, the cell proliferation inhibition rates of the three experimental groups were significantly higher than those of the control group （ P 〈 0.05 ）, and the differences between each two experimental groups were statistically significant （ P 〈 0.05 ）. The apoptosis rates of the three experimental groups were significantly higher than those of the control group （ P 〈 0.05 ）, and the differences between each two experimental groups were statistically significant （ P 〈 0.05 ）. Compared with the con- trol group, the expressions of AKT, roTOR and Bcl -2 mRNA of the three experimental groups were significantly decreased （P 〈 0.05 ） , and the expressions of Ban and Caspase - 3 mRNA were increased significantly （ P 〈 0.05 ） in the three experimental groups at 48h, of which the expressions of the proteins of Caspase3, Bax and Bcl -2 were consistent with the mRNA expressions. Conclusion：cinobufagin can inhibit pro-liferation and promote apoptosis of hepatoma cell line, and it suggests that the down - regulation of Bel - 2 and up - regulation of Bax and Caspase -3 can inhibit the proliferation and promote apoptosis of HCC cells ,which is related to AKT/mTOR signaling pathway.