摘要
目的研究自噬在风车子抑素A4(Combretastatin A4,CA4)抑制人脐静脉内皮细胞(HUVECs)增殖中的作用,探讨CA4抑制血管生成的机制。方法体外培养HUVECs,采用不同浓度的CA4处理HUVECs,药物作用不同时间后用MTT法检测CA4对细胞存活率的影响,MDC染色检测10n M CA4处理不同时间自噬小体变化的情况,RT-PCR检测自噬相关基因LC3B和Beclin 1基因转录水平的变化,Western Blot检测LC3B和Beclin1的蛋白表达水平的变化。结果 CA4能抑制HUVECs增殖并在一定范围呈浓度依赖性和时间依赖性;10n M CA4处理12小时和24小时自噬小体显著增加,诱导HUVECs自噬;荧光定量PCR结果显示自噬相关基因LC3Ⅰ表达下调、LC3Ⅱ表达上调,LC3Ⅰ/LC3Ⅱ比值显著上调,Beclin 1表达上调;Western Blot结果与荧光定量PCR结果一致,但m TOR蛋白未见明显变化。结论 CA4能显著抑制HUVECs增殖和诱导HUVECs自噬。
Objective To investigate the role of autophagy in the proliferation inhibition effect of Combretastatin A4( CA4) on human umbilical vein endothelial cells( HUVECs),and to explore the mechanism of angiogenesis inhibition. Methods HUVECs were cultured in vitro and treated with different concentration of CA4.The effect of CA4 on cell survival rate was determined by MTT method after a varying period of time,the treatment effect of 10 n M CA4 on autophagosome at different time was determined by Monodansylcadaverin( MDC)staining methods,the genetic transcription level changes of autophagy-associated genes,LC3 and Beclin 1,were determined by Real Time PCR,the protein expression level changes of LC3 and Beclin 1 were detected by Western Blot. Results CA4 can inhibit the proliferation of HUVECs,the inhibition depends on concentration and time in certain range,the autophagosomes treated by 10 n M CA4 for 12 hours and 24 hours increased significantly,inducing autophagy in HUVECs cells,the results of real time PCR showed that the expression of LC3Ⅰ,the autophagy-related gene,was down-regulated,the expression of LC3 Ⅱ was up-regulated,the LC3Ⅰ to LC3Ⅱ ratio was significantly up-regulated and the expression of Beclin 1 was up-regulated,the results of Western Blot was the same as that of real time PCR,but there was no obvious changes in the expression of m TOR protein.Conclusion CA4 can inhibit the proliferation of HUVECs significantly and induce the autophagy of HUVECs.
出处
《邵阳学院学报（自然科学版）》
2017年第6期90-96,共7页
Journal of Shaoyang University：Natural Science Edition
基金
湖南省教育厅科学研究项目(15C1465)
湖南省教育厅优秀青年项目(15B220)
关键词
风车子抑素A4
自噬
内皮细胞
combretastatin A4
autophagy
endothelial cells
