【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。更多还原

【Abstract】 AIM: To investigate the effect of ethyl docosahexaenoate( Et-DHA) on the apoptosis of human hepatocarcinoma 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 MTT. Nuclear morphological features of the HepG2 cells were observed 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 cells,the effects of proliferation of Et-DHA-treated T cells on the activity of HepG2 cells were observed,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-treated 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 levels decreased,and the cytoplasmic Cyt C,Smac,cleaved caspase-8,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.更多还原