【摘要】：目的： 觀察自噬對氧化低密度脂蛋白（ox-LDL）在人臍靜脈內(nèi)皮細(xì)胞（HUVECs）中聚集的影響。 方法： 1.運用不同時間點的Dil-ox-LDL處理HUVECs，采用流式細(xì)胞技術(shù)檢測HUVECs中Dil-ox-LDL的含量。 2.在自噬誘導(dǎo)劑雷帕霉素和抑制劑3甲基腺嘌呤（3MA）干預(yù)下，觀察自噬對Dil-ox-LDL在HUVECs中聚集的影響。 3.在雷帕霉素和3MA干預(yù)下，通過免疫熒光技術(shù)觀察HUVECs中Dil-ox-LDL與自噬標(biāo)記物L(fēng)C3以及溶酶體標(biāo)記物L(fēng)AMP1的共定位情況，，以及HUVECs中LC3與LAMP1的共定位情況，初步探討自噬抑制HUVECs中ox-LDL聚集的可能機制；采用蛋白免疫印跡方法檢測自噬流相關(guān)蛋白LC3、beclin1、LAMP1、P62的蛋白含量，進(jìn)一步證實自噬參與了ox-LDL在HUVECs中的降解。 結(jié)果： 1.流式細(xì)胞術(shù)檢測結(jié)果顯示Dil-ox-LDL在HUVECs中的積聚量隨時間點的延長而增加，3h細(xì)胞內(nèi)Dil-ox-LDL是0小時的8.53倍，3h時間點下雷帕霉素可抑制HUVECs中ox-LDL的積聚（P=0.003）而3MA能夠增加HUVECs中ox-LDL的積聚（P=0.035）。 2.免疫熒光技術(shù)顯示3h時間點下，Dil-ox-LDL與LC3及LAMP1發(fā)生大量共定位，此時LC3與LAMP1也大量共定位，同時Dil-ox-LDL與LC3及LAMP1、LC3與LAMP1的共定位能夠被雷帕霉素增加，被3MA所抑制。 3.免疫印跡技術(shù)結(jié)果顯示，ox-LDL作用于HUVECs3h后，LC3-II/LC3-I（P=0.024）、beclin1（P=0.005）及LAMP1（P＜0.001）蛋白表達(dá)明顯增加，P62蛋白水平降低（P＜0.001）；與ox-LDL組相比雷帕霉素能夠進(jìn)一步增加LC3-II/LC3-I（P=0.045）和beclin1（P=0.001）蛋白表達(dá)，降低P62（P=0.030）蛋白表達(dá)；與ox-LDL組相比3MA能夠降低LC3-II/LC3-I（P＜0.001）和beclin1（P＜0.001）蛋白表達(dá)，增加P62（P＜0.001）蛋白表達(dá)。 結(jié)論： ox-LDL在HUVECs中聚集隨著作用時間的延長而增加，雷帕霉素作用后可抑制ox-LDL在HUVECs中聚集，雷帕霉素上調(diào)ox-LDL作用后HUVECs中自噬流水平，增高的自噬流促進(jìn)自噬泡對ox-LDL吞噬，進(jìn)一步和溶酶體融合后，在自噬溶酶體中降解，自噬溶酶體通路參與HUVECs中ox-LDL的降解。
[Abstract]:Aim: to observe the effect of autophagy on the aggregation of oxidized low density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs). Methods: 1. The content of Dil-ox-LDL in HUVECs was detected by flow cytometry with HUVECs, treated with Dil-ox-LDL at different time points. 2. 2. The effects of autophagy on the aggregation of Dil-ox-LDL in HUVECs were observed under the intervention of rapamycin and 3-methyladenine (3MA). Under the intervention of rapamycin and 3MA, the co-localization of Dil-ox-LDL and autophagy LC3 and lysosomal marker LAMP1 in HUVECs and LC3 and LAMP1 in HUVECs were observed by immunofluorescence technique. To explore the possible mechanism of autophagy inhibiting ox-LDL aggregation in HUVECs and to detect the protein content of autophagy associated protein LC3,beclin1,LAMP1,P62 by Western blot, it was further proved that autophagy was involved in the degradation of ox-LDL in HUVECs. Results: 1. The results of flow cytometry showed that the accumulation of Dil-ox-LDL in HUVECs increased with the prolongation of time point. The intracellular Dil-ox-LDL in 3 h was 8.53 times higher than that in 0 h. Rapamycin inhibited the accumulation of ox-LDL in HUVECs (P0. 003) at 3 h, and 3MA increased ox-LDL in HUVECs. Accumulation (P0. 035). Immunofluorescence technique showed that there was a large number of co-localization between Dil-ox-LDL and LC3 and LAMP1 at the time of 3 h, LC3 and LAMP1 were also co-located, and the co-localization of Dil-ox-LDL and LC3, LAMP1,LC3 and LAMP1 could be increased by rapamycin and inhibited by 3MA. The results of Western blotting showed that the expression of LC3-II/LC3-I (P0. 024), beclin1 (P0. 005) and LAMP1 (P < 0.001) and the level of P62 protein decreased (P < 0.001) after ox-LDL treatment on HUVECs3h, and rapamycin could further increase the expression of LC3-II/LC3-I (Pn0. 045) and beclin1 (Pn0. 001) protein and decrease the expression of P62 (P0. 030) protein compared with ox-LDL group. Compared with ox-LDL group, 3MA decreased the expression of LC3-II/LC3-I (P < 0.001) and beclin1 (P < 0.001), and increased the expression of P62 (P < 0.001). Conclusion: the aggregation of ox-LDL in HUVECs increases with the prolongation of action time. Rapamycin can inhibit the aggregation of ox-LDL in HUVECs, and rapamycin upregulate the level of autophagy in HUVECs after ox-LDL treatment. The increased autophagy promoted the phagocytosis of ox-LDL by autophagy, and after further fusion with lysosome, the autophagy pathway was involved in the degradation of ox-LDL in HUVECs.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363

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