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目的:研究二氯化钴(CoCl_2)对唾液腺腺样囊性癌ACC-M细胞增殖和自噬的影响。方法:应用MTT法对ACC-M细胞进行细胞活力测验,以检测CoCl_2对细胞增殖的抑制作用;透射电镜观察自噬体形成;双免疫荧光标记、实时定量PCR(RT-PCR)及Western蛋白免疫印迹检测自噬相关基因HIF-1α/BNIP3通路相关基因、自噬相关蛋白微管相关蛋白1轻链3(microtubule associated protein 1 light chain 3,LC3)及Beclin 1的表达。采用SPSS15.0软件包对数据进行双尾t检验。结果:CoCl_2可降低ACC-M细胞的活性,同时诱导自噬体的形成;透射电镜下,实验组细胞内可见自噬体样的双层膜结构;RT-PCR及Western蛋白免疫印迹检测显示,CoCl_2可使HIF-1α/BNIP3信号通路和自噬相关蛋白的表达显著提高。结论:CoCl_2可模拟低氧环境,从而诱导ACC-M细胞产生自噬,HIF-1α/BNIP3信号通路在这一过程中扮演着重要角色。
Objective: To study the effects of cobalt chloride (CoCl_2) on the proliferation and autophagy of salivary adenoid cystic carcinoma ACC-M cells. Methods: Cell viability assay was performed on ACC-M cells by MTT to detect the inhibitory effect of CoCl 2 on cell proliferation. Transmission electron microscopy was used to observe the formation of autophagosomes. Double immunofluorescence labeling, real-time quantitative PCR (RT-PCR) and Western protein immunization Western blotting was used to detect the expression of HIF-1α / BNIP3 pathway related genes, microtubule associated protein 1 light chain 3 (LC3) and Beclin 1 in autophagy-related genes. Two-tailed t-test was performed on the data using SPSS15.0 software package. Results: CoCl 2 could reduce the activity of ACC-M cells and induce the formation of autophagosomes. Under the transmission electron microscope, autophagosome-like bilayer membrane was observed in the cells of experimental group. RT-PCR and Western blotting showed that, CoCl 2 significantly increased the expression of HIF-1α / BNIP3 signal pathway and autophagy-related protein. CONCLUSION: CoCl 2 can mimic the hypoxic environment and induce autophagy in ACC-M cells. HIF-1α / BNIP3 signaling plays an important role in this process.