雷帕霉素诱导人黑素瘤细胞M14自噬及Bcl-2?Bax表达的变化

目的:对雷帕霉素诱导黑素瘤细胞发生自噬的过程进行观察,并初步探讨其可能的机制?方法:不同浓度雷帕霉素(10?50?100 nmol/L)处理黑素瘤细胞M14,采用MDC荧光染色检测自噬囊泡;免疫细胞化学法检测自噬蛋白LC3B的表达;透射电子显微镜观察黑素瘤细胞超微结构的变化;Western blot检测细胞内凋亡相关蛋白Bcl-2和Bax的表达;采用罗丹明123染色(Rhodamine 123)流式细胞术检测线粒体膜电位的变化;MTT比色法检测雷帕霉素对M14细胞增殖的抑制作用?结果:经不同浓度的雷帕霉素处理后,MDC阳性细胞数增多,M14细胞发生自噬;自噬蛋白LC3B的表达增强;自噬水平随雷帕霉素浓度的升高而逐渐增强?透射电子显微镜观察可见M14细胞质内有大量独立双层膜结构?线粒体肿胀并出现自噬体?自噬溶酶体?Western blot结果显示雷帕霉素可以下调M14细胞中的凋亡抑制蛋白Bcl-2的表达,同时上调凋亡诱导蛋白Bax的表达?雷帕霉素可引起M14细胞的线粒体膜电位下降,与对照组相比差异显著(P < 0.05)?10~100 nmol/L的雷帕霉素明显抑制黑素瘤细胞M14的增殖,该作用呈剂量依赖性,随着药物浓度的升高,抑制率增加,与空白对照组比较均有显著性差异(P < 0.01)?结论:10~100 nmol/L的雷帕霉素可诱导黑素瘤细胞发生自噬,抑制细胞的生长?其机制可能与蛋白Bcl-2和Bax表达水平有关?

Study on autophagy induced by rapamycin in melanoma cell line M14 and changes in the expression of Bcl-2 and Bax in progress of autophagy

Objective:To observe the autophagy of melanoma cell induced by rapamycin and explore the possible mechanism. Methods: M14 cells were cultured in vitro in the presence of rapamycin at three concentrations(10,50,100 nmol/L) for 24 hours. MDC fluorescent staining was used to detect autophagic vesicles. Immunocytochemistry was used to detect the expression of autophagy protein LC3B. Autophagosome was observed under transmission electronic microscopy. The expression levels of apoptosis-related protein Bcl-2 and Bax were measured by Western blot. Changes of mitochondrial membrane potential were examined by Rhodamine 123 dye and flow cytometry. The effects of rapamycin on proliferation of M14 cells were assessed using the MTT assay. Results: After rapamycin treatment,autophagy was induced in M14 cells as detected by MDC staining. The expression of LC3B increased. The intensity of autophagy was correlated with the concentration of rapamycin. Independent double membranes,swelling mitochondrias,autophagosomes and autolysosomes were observed in rapamycin-treated M14 cells under transmission electronic microscopy. Western blot revealed an upregulated expression of Bcl-2 and downregulated expression of Bax. Rapamycin at the concentrations of 10 to 100 nmol/L could markedly reduce the mitochondrial membrane potential compared with the control group(P < 0.05),and inhibit the proliferation of M14 cells in a dose-dependent manner. The growth inhibition was significantly higher than that of the control group(P < 0.01). Conclusion: Rapamycin at the concentrations of 10 to 100 nmol/L could induce autophagy in melanoma cells,and inhibit the growth of melanoma cells. The mechanism may be associated with the expression level of protein Bcl-2 and Bax.