摘要
目的:本研究探讨了二十二碳六烯酸乙酯(Et-DHA)对人肝癌HepG2细胞凋亡的影响。方法:HepG2细胞用于检测Et-DHA的抑癌活性,MTT法检测Et-DHA对HepG2细胞的直接抑制作用,Hoechst 33258荧光染色观察细胞的形态特征,ELISA法检测Et-DHA处理后HepG2细胞的活性氧簇(ROS)释放量、总超氧化物歧化酶(SOD)和caspase-9活性,Western blotting法检测胞质和线粒体中Bax、Bak、Bid、Bcl-2、Smac和细胞色素C(Cyt C),以及胞质中cleaved caspase-8、cleaved caspase-9和cleaved caspase-3的水平;T细胞与HepG2细胞共培养,进一步观察Et-DHA处理后T细胞的增殖对HepG2细胞活性的影响,并检测了颗粒酶(granzyme)B的水平。结果:Et-DHA显著抑制HepG2细胞的生长(P<0.05),这种抑制作用具浓度效应和时程效应;Et-DHA处理后HepG2细胞的ROS释放量增加,但总SOD活性无明显变化,caspase-9活性显著上升(P<0.05);线粒体上的促凋亡蛋白Bax、Bak和Bid水平增加,而抑凋亡蛋白Bcl-2以及线粒体中Cyt C和Smac的水平降低,胞质中的Cyt C、Smac、cleaved caspase-8、cleaved caspase-9、cleaved caspase-3以及cleaved Bid水平呈剂量性升高。另外T细胞和HepG2细胞共培养组在Et-DHA的诱导下,HepG2细胞的凋亡程度与Et-DHA单独作用时相比进一步增加。在Et-DHA刺激下,T细胞内granzyme B上调,释放到HepG2细胞内的granzyme B明显增多。结论:Et-DHA可能主要通过线粒体内源性途径以及caspase-8途径,激活caspase-3,诱导HepG2细胞凋亡,以及通过间接活化T细胞,促使granzyme B增多,从而增强对HepG2细胞的毒性作用。
AIM： To investigate the effect of ethyl docosahexaenoate （Et-DHA） on the apoptosis of human hepatocarcin0ma HepG2 cells. METHODS ： HepG2 cells were used to test the anticarcinogenicity of Et-DHA. The direct inhibition of HepG2 cells by Et-DHA was detected by MrIT. Nuclear morphological features of the HepG2 ceils were ob served under fluorescence microscope after staining with Hochest 33258. The levels of Bax, Bak, Bid, Bcl-2, Smac and cytochrome C （Cyt C） in mitochondria and cytosol, the cleaved caspase-8, cleaved caspase-9, and cleaved caspase-3 in cytosol, as well as the release of reactive oxygen species （ ROS）, total superoxide dismutase （SOD） and caspase-9 activity in the Et-DHA-treated HepG2 cells were determined by Western blotting and ELISA. Furthermore, by co-culturing the HepG2 cells with T ceils, the effects of proliferation of Et-DHA-treated T cells on the activity of HepG2 cells were ob served, and the level of granzyme B was detected. RESULTS ： Et-DHA significantly inhibited the growth of HepG2 cells ina concentration and time-dependent manner. The ROS release and caspase-9 activity increased markedly in Et-DHA-trea- ted HepG2 cells, and no significant change of the total SOD activity was observed. The levels of the pro-apoptotic proteins Bax, Bak and Bid in mitochondria increased, the anti-apoptotic protein Bcl-2 as well as mitochondrial Cyt C and Smac lev els decreased, and the cytoplasmic Cyt C, Smac, cleaved caspase-$, cleaved caspase-9, cleaved caspase-3 and cleaved Bid levels showed dose-dependent increases. Additionally, the degree of Et-DHA-induced apoptosis in HepG2 cells in the co-culture group （ T cells ＋ HepG2 cells） showed a further increase as compared with the HepG2 cells treated with Et-DHA alone. Due to Et-DHA inducing elevation of granzyme B level in the T cells, the granzyme B released into HepG2 cells was significantly increased. CONCLUSION ： Et-DHA might induce the apoptosis of HepG2 cells through activation of caspase 3 mainly via a mitochondrial intrinsic pathway and a caspase-8 pathway, and promote the increase in granzyme B indirectly by activating T cells, thus enhancing the cytotoxic effect on HepG2 cells.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2014年第3期385-393,共9页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.31171281)
广州市教育局科技计划项目(No.10A044)
