Ox-LDL对THP-1巨噬细胞自噬的影响及其作用机制的初步研究

发布时间：2018-03-23 21:44

本文选题：Ox-LDL　切入点：THP-1源性巨噬细胞　出处：《南华大学》2013年硕士论文

【摘要】：目的：氧化低密度脂蛋白(oxdized low density lipoprotein, Ox-LDL)是低密度脂蛋白（low density lipoprotein,LDL）被活性氧（ROS）或者二价金属离子（Cu~(2+)，Fe~(2+)）氧化的产物。巨噬细胞通过清道夫受体吞噬Ox-LDL，，转化为巨噬细胞源性泡沫细胞，介导动脉粥样硬化（Atherosclerosis, As）的形成和发展。近年来的研究发现，动脉粥样硬化斑块中存在巨噬细胞自噬，但Ox-LDL是否调节巨噬细胞自噬及其调节机制尚不清楚。方法：1. PMA诱导THP-1细胞24h，使分化为巨噬细胞，再分别用0，10，20，40，80mg/L的Ox-LDL处理24h，逆转录聚合酶链反应（RT-PCR）和免疫印迹技术（Western blot）分别检测LC3、Beclin1、TET2的mRNA及蛋白表达情况；细胞免疫荧光检测LC3在细胞内的表达情况；MDC染色检测自噬囊泡的形成。 2.TET2siRNA转染或DNA甲基化抑制剂(5-Aza-2’-deoxycytidine,5-aza-CdR)处理THP-1源性巨细胞24h，再用80mg/L的Ox-LDL处理细胞24h，RT-PCR和WesternBlot分别检测LC3、Beclin1、TET2的mRNA及蛋白的表达情况细胞免疫荧光检测细胞内LC3的表达；MDC染色检测自噬囊泡形成。结果： 1.随着Ox-LDL浓度的增加，THP-1巨噬细胞Beclin1mRNA及蛋白表达显著降低（P0.05）；LC3mRNA表达无明显改变（P0.05）,但其蛋白表达显著减少（P0.001）；细胞免疫荧光结果表明随Ox-LDL浓度的增加，LC3含量降低，MDC染色结果显示随着Ox-LDL的浓度增加，自噬囊泡减少。 2.TET2siRNA转染或DNA甲基化抑制剂处理THP-1巨噬细胞，实验结果显示：与80mg/L组相比，TET2siRNA组Beclin1mRNA及蛋白表达明显下调（P㩳0.01），但AzadC组Beclin1mRNA及蛋白表达明显上调；TET2siRNA组LC3的mRNA及蛋白表达水平明显降低，但AzadC组LC3的mRNA及蛋白表达水平增加；MDC染色显示TET2siRNA转染组的自噬水平明显降低；细胞免疫荧光显示TET2siRNA转染后，LC3在细胞内的含量降低。结论：Ox-LDL通过浓度依赖性下调TET2的表达抑制THP-1源性巨噬细胞自噬。
[Abstract]:Objective: oxidized low density lipoprotein (Ox-LDL) is the product of oxidizing low density lipoprotein density lipoprotein (LDLs) by reactive oxygen species (Ros) or divalent metal ions (CU). Macrophages engulf Ox-LDLthrough scavenger receptors and are transformed into macrophage-derived foam cells. Recent studies have found that macrophage autophagy exists in atherosclerotic plaques, but it is not clear whether Ox-LDL regulates macrophage autophagy and its regulatory mechanism. Methods 1. PMA induced THP-1 cells to differentiate into macrophages for 24 h, and then treated with Ox-LDL for 24 h. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression of mRNA and protein in LC3 / Beclin1 and TET2, respectively. The expression of LC3 in cells was detected by immunofluorescence staining and the formation of autophagic vesicles was detected by MDC staining. THP-1 derived giant cells were treated with 2.TET2siRNA transfection or DNA methylation inhibitor (5-Aza-2) -deoxycytidine (5-aza-CdR) for 24 h, followed by 80mg/L Ox-LDL treatment for 24 h, RT-PCR and WesternBlot were used to detect the expression of mRNA and protein in LC3Beclin1 + TET2, respectively. The expression of LC3 in the cells was detected by MDC staining for the formation of autophagic vesicles. Results:. 1. With the increase of Ox-LDL concentration, the expression of Beclin1mRNA and protein in THP-1 macrophages did not change significantly, but its protein expression decreased significantly with the increase of Ox-LDL concentration. The fruit showed that as the concentration of Ox-LDL increased, Autophagy vesicles decreased. THP-1 macrophages were treated with 2.TET2siRNA transfection or DNA methylation inhibitor. The results showed that the expression of Beclin1mRNA and protein in Tet 2 siRNA group was significantly lower than that in 80mg/L group. However, the expression of mRNA and protein in LC3 of AzadC group decreased significantly, but the mRNA and protein expression level of LC3 in AzadC group increased and the autophagy level of TET2siRNA transfected group decreased significantly. Cell immunofluorescence showed that the content of LC3 decreased after TET2siRNA transfection. Conclusion the TET2 expression was down-regulated in a dose-dependent manner by% Ox-LDL to inhibit THP-1 derived macrophage autophagy.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R543.5

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