自噬抑制氧化低密度脂蛋白在人血管内皮细胞聚集的作用及机制探讨
发布时间：2018-10-22 20:44
【摘要】：目的： 观察自噬对氧化低密度脂蛋白（ox-LDL）在人脐静脉内皮细胞（HUVECs）中聚集的影响。 方法： 1.运用不同时间点的Dil-ox-LDL处理HUVECs，采用流式细胞技术检测HUVECs中Dil-ox-LDL的含量。 2.在自噬诱导剂雷帕霉素和抑制剂3甲基腺嘌呤（3MA）干预下，观察自噬对Dil-ox-LDL在HUVECs中聚集的影响。 3.在雷帕霉素和3MA干预下，通过免疫荧光技术观察HUVECs中Dil-ox-LDL与自噬标记物LC3以及溶酶体标记物LAMP1的共定位情况，，以及HUVECs中LC3与LAMP1的共定位情况，初步探讨自噬抑制HUVECs中ox-LDL聚集的可能机制；采用蛋白免疫印迹方法检测自噬流相关蛋白LC3、beclin1、LAMP1、P62的蛋白含量，进一步证实自噬参与了ox-LDL在HUVECs中的降解。 结果： 1.流式细胞术检测结果显示Dil-ox-LDL在HUVECs中的积聚量随时间点的延长而增加，3h细胞内Dil-ox-LDL是0小时的8.53倍，3h时间点下雷帕霉素可抑制HUVECs中ox-LDL的积聚（P=0.003）而3MA能够增加HUVECs中ox-LDL的积聚（P=0.035）。 2.免疫荧光技术显示3h时间点下，Dil-ox-LDL与LC3及LAMP1发生大量共定位，此时LC3与LAMP1也大量共定位，同时Dil-ox-LDL与LC3及LAMP1、LC3与LAMP1的共定位能够被雷帕霉素增加，被3MA所抑制。 3.免疫印迹技术结果显示，ox-LDL作用于HUVECs3h后，LC3-II/LC3-I（P=0.024）、beclin1（P=0.005）及LAMP1（P＜0.001）蛋白表达明显增加，P62蛋白水平降低（P＜0.001）；与ox-LDL组相比雷帕霉素能够进一步增加LC3-II/LC3-I（P=0.045）和beclin1（P=0.001）蛋白表达，降低P62（P=0.030）蛋白表达；与ox-LDL组相比3MA能够降低LC3-II/LC3-I（P＜0.001）和beclin1（P＜0.001）蛋白表达，增加P62（P＜0.001）蛋白表达。 结论： ox-LDL在HUVECs中聚集随着作用时间的延长而增加，雷帕霉素作用后可抑制ox-LDL在HUVECs中聚集，雷帕霉素上调ox-LDL作用后HUVECs中自噬流水平，增高的自噬流促进自噬泡对ox-LDL吞噬，进一步和溶酶体融合后，在自噬溶酶体中降解，自噬溶酶体通路参与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.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
本文编号：2288260
[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.
【学位授予单位】：苏州大学
【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4条
1 莫中成;易光辉;陈欣;唐朝克;王佐;刘录山;杨永宗;;氧化低密度脂蛋白对THP-1巨噬细胞CD36表达的影响[J];医学研究生学报;2006年01期
2 严鹏科,廖端芳,杨永宗;Caveolin-1表达对血管平滑肌细胞胆固醇逆转运的调节作用[J];中国动脉硬化杂志;2002年05期
3 陈玉成,梁玉佳,刘瑞,黄明慧,曾智;氧化低密度脂蛋白对血管内皮细胞骨架的损伤及其机制[J];心血管康复医学杂志;2004年04期
4 朱冰坡,范利;动脉粥样硬化的抗氧化治疗研究进展[J];中国老年学杂志;2005年04期
本文编号：2288260
本文链接：https://www.wllwen.com/xiyixuelunwen/2288260.html
最近更新
教材专著
