自噬在熊果酸抑制血管内皮细胞增殖中的作用

发布时间：2018-08-02 18:02

【摘要】：目的：研究自噬在熊果酸（Ursolic acid，UA）抑制人脐静脉血管内皮细胞（human umbilical vein endothelial cells，HUVECs）增殖中的作用。 方法：体外培养HUVECs，采用不同浓度的熊果酸和自噬特异性抑制剂3-甲基腺嘌呤（3-methyladenine，3-MA,）与熊果酸联合处理。运用MTT法检测熊果酸对HUVECs增殖的抑制作用及其联合3-MA处理细胞后细胞存活状态的变化；在透射电镜下观察熊果酸作用前后HUVECs结构的改变；微管相关蛋白1轻链3（microtubule-associatedprotein1light chain3, LC3）免疫荧光和流式细胞术检测熊果酸对HUVECs自噬水平的影响；单丹磺酰戊二胺（monodansylcadaverine，MDC）荧光染色法观察熊果酸对HUVECs自噬泡的影响；Westernblotting检测LC3及自噬相关蛋白Beclin-1蛋白的表达情况；RT-PCR检测LC3及Beclin-1转录水平的变化；流式细胞术检测HUVECs经熊果酸和3-MA与熊果酸联合处理后凋亡率的变化。 结果：随着熊果酸作用于HUVECs剂量的增加细胞的生长抑制程度及死亡程度也逐渐的增强；熊果酸抑制HUVECs生长增值36h的半数作用剂量为20μmol/L；HUVECs经熊果酸处理后透射电镜及MDC染色均显示:细胞浆中自噬相关形态的数量具有明显上升的趋势;细胞免疫荧光法检测显示熊果酸处理HUVECs组LC3阳性细胞数较对照组明显增强且流式细胞术检测显示熊果酸处理HUVECs后细胞的荧光强度与正常细胞培养组相比明显增强; HUVECs经熊果酸处理后可上调LC3及Beclin-1的蛋白水平及mRNA的转录水平；3-MA与熊果酸联合作用后，HUVECs受到的增殖抑制明显高于熊果酸单独作用，且其凋亡率上升明显。 结论：熊果酸抑制人脐静脉血管内皮细胞生长增殖，，在这个过程中自噬现象被激活，自噬在此过程中对HUVECs起保护作用。自噬抑制剂3-MA能够增强熊果酸抑制HUVECs生长增殖的作用并且促进熊果酸诱导HUVECs发生凋亡。
[Abstract]:Aim: to investigate the role of autophagy in inhibiting the proliferation of human umbilical vein endothelial cells (human umbilical vein endothelial cells HUVECs) induced by Ursolic acidinic acid (UA). Methods: HUVECs were cultured in vitro and treated with different concentrations of ursolic acid and 3-methyladenine 3-MA (3-methyladenine 3-MA) in combination with ursolic acid. The inhibitory effect of ursolic acid on the proliferation of HUVECs and the changes of cell viability after treated with ursolic acid and 3-MA were detected by MTT method, and the changes of HUVECs structure before and after ursolic acid treatment were observed under transmission electron microscope. The effect of ursolic acid on HUVECs autophagy was detected by microtubule-associatedprotein1light chain 3 (LC3) immunofluorescence and flow cytometry, and the effect of ursolic acid on HUVECs autophagy was observed by monodactyl succinylenediamine (Monodanyl succinylenediamine) fluorescence staining. Westernblotting was used to detect the expression of LC3 and Beclin-1 protein, RT-PCR was used to detect the changes of LC3 and Beclin-1 transcription, and flow cytometry was used to detect the apoptosis rate of HUVECs treated with ursolic acid, 3-MA and ursolic acid. Results: with the increase of ursolic acid dose on HUVECs, the cell growth inhibition and death degree increased gradually, and the half dose of ursolic acid inhibited the growth and increment of HUVECs for 36 h was 20 渭 mol 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1); Transmission electron microscopy (TEM) and MDC staining of HUVECs treated with ursolic acid showed that the number of autophagy related forms in the cytoplasm was obviously increasing. Cell immunofluorescence assay showed that the number of LC3 positive cells in ursolic acid treated HUVECs group was significantly higher than that in control group. Flow cytometry showed that the fluorescence intensity of ursolic acid treated HUVECs cells was significantly higher than that of normal cell culture group. The protein levels of LC3 and Beclin-1 and the transcription level of mRNA were up-regulated by ursolic acid treatment with HUVECs. The proliferation inhibition of 3-MA combined with ursolic acid was significantly higher than that of ursolic acid alone, and the apoptosis rate of HUVECs increased significantly. Conclusion: ursolic acid inhibits the growth and proliferation of human umbilical vein endothelial cells, during which autophagy is activated, and autophagy plays a protective role on HUVECs during this process. Autophagy inhibitor 3-MA could enhance the inhibitory effect of ursolic acid on the growth and proliferation of HUVECs and promote the apoptosis of HUVECs induced by ursolic acid.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

【参考文献】

相关期刊论文 前8条

1 李国东;吴德全;李本义;;细胞自噬在肿瘤中作用的研究进展[J];癌症;2009年04期

2 张文娟;陈少鹏;陆鑫;周国春;;熊果酸衍生物的合成及抗肿瘤血管生成活性[J];高等学校化学学报;2010年11期

3 伍静;赵勇;师长宏;张海;;自噬的形态特征及分子调控[J];现代生物医学进展;2010年20期

4 李伟;刘琼;何林;毕娟娟;叶秀峰;;熊果酸对血管内皮细胞增殖的影响及其机制[J];中国生物制品学杂志;2011年02期

5 赵黎明;徐寒梅;奚涛;;肿瘤血管生成及其抑制剂研究进展[J];药物生物技术;2010年05期

6 孔晓霞;张宏宇;钟加滕;康劲松;孙连坤;;自噬抑制剂3-甲基腺嘌呤在H_2O_2引起的神经胶质瘤U251细胞损伤中的作用[J];中国病理生理杂志;2011年04期

7 李颖;郑毅涛;齐一鸣;黄俊琪;;登革病毒2型诱导人血管内皮细胞株EAhy926自噬的研究[J];中山大学学报(医学科学版);2011年04期

8 齐亚莉;王俊;李岩;王洪艳;龚守良;;自噬体的检测及其对肿瘤的作用[J];中国实验诊断学;2010年05期

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