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目的研究自噬在熊果酸(Ursolic acid,UA)抑制人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)增殖中的作用,探讨UA抑制血管生长的机制。方法体外培养HUVECs,分别采用不同浓度的UA和自噬特异性抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)+UA联合处理,采用MTT法检测UA对HUVECs增殖的抑制作用及其联合3-MA对HUVECs存活率的影响;透射电镜观察细胞的超微结构;微管相关蛋白1轻链3(Microtubule-associated protein 1 light chain 3,MAP1-LC3)免疫荧光和流式细胞术检测UA对HUVECs自噬表达水平的影响;RT-PCR检测自噬相关基因Beclin1和MAP1-LC3B转录水平的变化。结果 UA可抑制HUVECs增殖,且呈剂量依赖性;HUVECs经UA处理后,自噬泡数量明显增加;经UA处理后,可上调HUVECs中MAP1-LC3蛋白的表达水平,MAP1-LC3阳性细胞的荧光强度较对照组明显增强;UA处理HUVECs不同时间可上调MAP1-LC3B及Beclin1 mRNA的转录水平;而UA联合3-MA处理后,HUVECs的增殖抑制作用明显增强。结论UA抑制HUVECs增殖,并诱导其发生自噬,自噬在此过程中起保护性作用,抑制自噬可明显增强UA诱导的HUVECs死亡。
Objective To investigate the role of autophagy in the proliferation of human umbilical vein endothelial cells (UUVECs) induced by Ursolic acid (UA), and to explore the mechanism of UA inhibiting the growth of human umbilical vein endothelial cells (HUVECs). Methods HUVECs were cultured in vitro and treated with different concentrations of UA and 3-methyladenine (3-MA) + UA, respectively. The inhibitory effect of UA on the proliferation of HUVECs was detected by MTT assay. The effect of 3-MA on the survival rate of HUVECs was observed. The ultrastructure of HUVECs was observed by transmission electron microscopy. The immunofluorescence and microtubule-associated protein 1 light chain 3 (MAP1-LC3) The effect of UA on the autophagy expression of HUVECs was examined. The changes of autophagy-related gene Beclin1 and MAP1-LC3B transcription were detected by RT-PCR. Results UA inhibited the proliferation of HUVECs in a dose-dependent manner. After treatment with UA, the number of autophagic vacuoles significantly increased. After treatment with UA, the expression of MAP1-LC3 protein and the fluorescence of MAP1-LC3 positive cells were increased Compared with the control group, UA treatment increased the transcriptional level of MAP1-LC3B and Beclin1 mRNA at different time points. However, UA combined with 3-MA significantly inhibited the proliferation of HUVECs. Conclusion UA can inhibit the proliferation of HUVECs and induce autophagy. Autophagy plays a protective role in this process. Inhibition of autophagy can significantly enhance the death of HUVECs induced by UA.