摘要
目的探讨乙醇对肝癌细胞凋亡的诱导作用及其与凋亡相关基因bax和bcl-2表达的关系。方法用噻唑蓝(MTT)法检测浓度分别为20～100 mL/ L的乙醇对肝癌细胞系HCC-9204的杀伤作用。然后，用60 mL/ L乙醇作用6 h诱导HCC-9204细胞凋亡。以May-Grunwald-Giemsa (MGG)染色法、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法和流式细胞术分别分析凋亡细胞的形态学变化、DNA断裂情况和不同细胞周期的DNA含量变化。用免疫细胞化学染色图象分析法，检测诱导凋亡前、后Bax和Bcl-2蛋白表达水平的变化。结果随着乙醇浓度的增加，其对HCC-9204细胞的杀伤作用逐渐增强。60 mL/ L乙醇可使HCC-9204细胞发生明显的细胞凋亡的形态学变化，并且大部分细胞为TUNEL阳性着色。流式细胞术分析可见明显的亚二倍体凋亡峰。诱导凋亡后的HCC-9204细胞Bax蛋白的表达水平明显增高，而Bcl-2在诱导凋亡前、后的HCC-9204细胞中均未见表达。结论低浓度乙醇对肝癌细胞HCC-9204具有明显的凋亡诱导作用，其凋亡的发生与Bax蛋白表达水平的增高有关。
Aim To explore the ethanol-induced apoptosis effect on hepatocellular carcinoma(HCC) cells and its relationship to the expression of apoptosis-associated genes, bax and bcl-2. Methods The cytotoxic effect of 20～100 mL/ L ethanol on HCC cell line HCC-9204 was tested by thiazolyl-blue (MTT) assay. Then apoptosis of HCC-9204 cells was induced with 60 mL/ L of ethanol for 6 h. The morphological change, DNA breakage and the change of DNA content of different cell cycles of the apoptotic cells were detected by May-Grunwald-Giemsa(MGG) staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and flow cytometer respectively. The changes of expression level of Bax and Bcl-2 proteins were detected by immunocytochemical staining and image analysis. Results The higher the concentration of ethanol was, the stronger the cytotoxic effect on HCC-9204 cells was. 60 mL/ L of ethanol could lead to obviously morphological apoptotic changes of HCC-9204 cells, and majority of the cells were TUNEL positive by TUNEL labeling assay. Typical apoptotic sub-G1 peak was observed by flow cytometer. The level of Bax protein expression increased significantly after induced with 60 mL/ L of ethanol for 6 h, no expression of Bcl-2 were found before and after induced with ethanol. Conclusion Low dose of ethanol can induce apoptosis of HCC-9204 cells obviously, and occurance of the apoptosis is related to the increase of the level of Bax protein expression.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2001年第4期315-317,358,共4页
Chinese Journal of Cellular and Molecular Immunology
