【摘要】：目的：研究自噬在熊果酸（Ursolic acid，UA）抑制人臍靜脈血管內(nèi)皮細(xì)胞（human umbilical vein endothelial cells，HUVECs）增殖中的作用。 方法：體外培養(yǎng)HUVECs，采用不同濃度的熊果酸和自噬特異性抑制劑3-甲基腺嘌呤（3-methyladenine，3-MA,）與熊果酸聯(lián)合處理。運(yùn)用MTT法檢測(cè)熊果酸對(duì)HUVECs增殖的抑制作用及其聯(lián)合3-MA處理細(xì)胞后細(xì)胞存活狀態(tài)的變化；在透射電鏡下觀察熊果酸作用前后HUVECs結(jié)構(gòu)的改變；微管相關(guān)蛋白1輕鏈3（microtubule-associatedprotein1light chain3, LC3）免疫熒光和流式細(xì)胞術(shù)檢測(cè)熊果酸對(duì)HUVECs自噬水平的影響；單丹磺酰戊二胺（monodansylcadaverine，MDC）熒光染色法觀察熊果酸對(duì)HUVECs自噬泡的影響；Westernblotting檢測(cè)LC3及自噬相關(guān)蛋白Beclin-1蛋白的表達(dá)情況；RT-PCR檢測(cè)LC3及Beclin-1轉(zhuǎn)錄水平的變化；流式細(xì)胞術(shù)檢測(cè)HUVECs經(jīng)熊果酸和3-MA與熊果酸聯(lián)合處理后凋亡率的變化。 結(jié)果：隨著熊果酸作用于HUVECs劑量的增加細(xì)胞的生長(zhǎng)抑制程度及死亡程度也逐漸的增強(qiáng)；熊果酸抑制HUVECs生長(zhǎng)增值36h的半數(shù)作用劑量為20μmol/L；HUVECs經(jīng)熊果酸處理后透射電鏡及MDC染色均顯示:細(xì)胞漿中自噬相關(guān)形態(tài)的數(shù)量具有明顯上升的趨勢(shì);細(xì)胞免疫熒光法檢測(cè)顯示熊果酸處理HUVECs組LC3陽(yáng)性細(xì)胞數(shù)較對(duì)照組明顯增強(qiáng)且流式細(xì)胞術(shù)檢測(cè)顯示熊果酸處理HUVECs后細(xì)胞的熒光強(qiáng)度與正常細(xì)胞培養(yǎng)組相比明顯增強(qiáng); HUVECs經(jīng)熊果酸處理后可上調(diào)LC3及Beclin-1的蛋白水平及mRNA的轉(zhuǎn)錄水平；3-MA與熊果酸聯(lián)合作用后，HUVECs受到的增殖抑制明顯高于熊果酸單獨(dú)作用，且其凋亡率上升明顯。 結(jié)論：熊果酸抑制人臍靜脈血管內(nèi)皮細(xì)胞生長(zhǎng)增殖，，在這個(gè)過(guò)程中自噬現(xiàn)象被激活，自噬在此過(guò)程中對(duì)HUVECs起保護(hù)作用。自噬抑制劑3-MA能夠增強(qiáng)熊果酸抑制HUVECs生長(zhǎng)增殖的作用并且促進(jìn)熊果酸誘導(dǎo)HUVECs發(fā)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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