HIV派生的microRNA99致巨噬细胞自噬的研究

发布时间：2018-06-05 01:19

  本文选题：miR99 + 巨噬细胞　； 参考：《山西医科大学》2017年硕士论文

【摘要】：目的:研究HIV派生的miR99能否引起巨噬细胞内自噬及对自噬功能的影响,从而进一步对HIV慢性免疫激活的机制进行探讨。方法:1.巨噬细胞获得:人单核细胞白血病细胞(THP-1)在佛波酯(PMA)刺激48小时后分化为贴壁巨噬细胞,倒置光学显微镜下观察细胞形态以鉴定。2.将巨噬细胞分为实验组(miR99组)、雷帕霉素组(RP组)、空白对照组,每组处置分别为加miR99、加雷帕霉素、不加任何试剂。每组均设置重复组。3.在各组处置后的第2、4、8、12、24小时等时间点取各组细胞裂解提取蛋白;重复组于8小时时刮取细胞,离心制细胞团保存于3%戊二醛内,交校内电镜教研室制标本。4.透射电镜下察看各组时间点自噬小体数并计数;Western blot方法检测自噬相关蛋白P62及LC3Ⅱ,Image J软件行半定量分析。5.收集资料,分析比较各组结果。结果:1.PMA刺激48小时后悬浮的THP-1细胞绝大部分转变为多边形的、贴壁巨噬细胞。2.透射电镜下观察,比之于空白对照组,RP组及miR99组自噬小体显著增多(P0.05),RP组及miR99组自噬小体相比无显著差异(P0.05)。3.目标蛋白的表达:(1)RP组:相较于空白对照组,LC3-Ⅱ的表达随时间(2h、4h、8h、12h、24h)递增(LC3-Ⅱ,P0.05);P62随时间(2h、4h、8h、12h、24h)递减(P62,P0.05)(2)mi R99组:相较于空白组,LC3Ⅱ的表达随时间(2h、4h、8h、12h、24h)递增(P0.05),P62于各时段几乎无变化(P0.05)。(3)miR99组与RP组相比较,LC3-Ⅱ的表达均随时间进展而递增,但miR99组的P62不随时间变化。结论:1.miR99引起巨噬细胞内自噬发生。2.miR99抑制巨噬细胞内自噬的降解功能。3.自噬体可能是巨噬细胞内HIV的储存库之一。4.对自噬功能的影响可能参与HIV慢性免疫激活的机制。
[Abstract]:Aim: to investigate whether miR99 derived from HIV can induce autophagy in macrophages and to explore the mechanism of chronic immune activation of HIV. Method 1: 1. Macrophages: human monocytic leukemia cells (THP-1) differentiated into adherent macrophages after 48 hours of stimulation by phorbol ester (PMA). The morphology of cells was observed under inverted optical microscope for identification of .2. The macrophages were divided into experimental group (n = 99), rapamycin group (n = 10) and blank control group (n = 10). Each group was treated with miR99 and rapamycin without any reagents. Each group was assigned to repeat group. 3. The cell lytic protein was obtained at 24 hours after treatment in each group, and the cells were scraped at 8 hours in the repeated group, and the centrifugal cell mass was preserved in 3% glutaraldehyde, and the samples were collected from the electron microscope teaching and research department in the school. The number of autophagy bodies and the number of autophagy bodies at each time point were examined under transmission electron microscope. Western blot method was used to detect autophagy associated protein P62 and LC3 鈪，

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