黄芩甙对肝癌细胞BEL-7402形态学的影响

EFFECT OF BAICALIN ON ULTRASTRUCTURE OF HCC CELL LINE BEL-7402

目的观察黄芩甙对肝癌细胞BEL-7402凋亡的影响,同时观察对肝癌细胞形态及超微结构、线粒体超微结构、线粒体膜电位和细胞内Ca2+的影响,探讨线粒体损伤在黄芩甙诱导肝癌细胞凋亡中的作用及可能的机制。方法应用细胞培养技术培养肝癌细胞BEL-7402,光镜、倒置显微镜、扫描电镜、透射电镜观察细胞形态及超微结构的变化尤其是线粒体的变化,应用流式细胞仪检测细胞凋亡百分率及线粒体膜电位、细胞内Ca2+的改变,免疫组化法检测细胞Bcl-2、Bax蛋白表达。结果黄芩甙诱导肝癌细胞BEL-7402凋亡呈剂量依赖关系,细胞形态、超微结构及线粒体超微结构出现明显改变,降低肝癌细胞线粒体膜电位,使细胞内Ca2+增加,细胞Bax表达增加,广泛分布于胞核和胞质中,Bcl-2表达减少。结论黄芩甙诱导肝癌细胞BEL-7402凋亡,线粒体损伤在黄芩甙诱导肝癌细胞凋亡中起重要作用,其机制可能为抑制肝癌细胞Bcl-2蛋白表达,促进Bax蛋白表达及细胞内Ca2+增加,激发线粒体膜通透性转运孔开放,线粒体跨膜电位降低,使肝癌细胞凋亡。

Objective To investigate the changes in morphology, ultrastructure, mitochondria membrane potential and intracellular Ca^2＋ of hepatocellular carcinoma （HCC） cell line BEL-7402 after treatment with baicalin, in order to explore the anti-tumour mechanism of baicalin. Methods HCC cell line BEL-7402 was cultured. The changes of morphology and ultrastructure were observed by light, inverted and electron microscopy. Mitochondria membrane potential, intracellular Ca^2＋ as well as apoptosis rate were determined by using flow cytometry, The expression of protein Bcl-2 and Bax was assessed by immunohistochemical technique. Results Baicalin induced BEL-7402 cell apoptosis in a concentration-dependent manner, as well as changes in morphology and ultrastructure. In the control group, the mitochondria membrane potential was stable and at high level, but in the treated group it was significantly decreased at 48h （P〈0. 01）. In the control group intracellular Ca^2＋ was changed little with time; in the baicalin treated group it was not different from that of the control group at 6h and 24h but significantly increased at 48h （P〈0.01）. The expression of bax was up-regulated, but the expression of bcl-2 was downregulated. Conclusion Baicalin induces apoptosis of hepatocarcinoma cell BEL-7402 in a dose dependent manner. Mitochondria play an important role in the apoptosis.