HIV派生的新型microRNA99对巨噬细胞TNF-α释放的影响及其机制

发布时间：2019-05-14 04:11

【摘要】：目的:探讨HIV派生的新型micro RNA99对巨噬细胞TNF-α释放的影响及其机制。方法:(1)构建巨噬细胞模型用PMA(佛波酯)刺激THP-1(人急性单核细胞白血病细胞)分化成巨噬细胞,于倒置显微镜下观察细胞形态是否发生变化。(2)HIV派生的新型micro RNA99对巨噬细胞TNF-α释放的影响以化学合成的物质作用于巨噬细胞,分组如下:(1)实验组1:巨噬细胞+转染试剂+micro RNA99(2)对照组1:巨噬细胞+转染试剂+micro RNA-TRA(3)对照组2:巨噬细胞+Lipid A(4)对照组3:巨噬细胞+培养基37℃、5%CO2的培养箱中培养24小时收集并检测上清液中TNF-α释放的情况。(3)HIV派生的新型micro RNA99对巨噬细胞TNF-α释放的机制研究ⅰTLR8质粒转染,实验分组如下:(1)实验组a:巨噬细胞+核转液+TLR8质粒(2)对照组b:巨噬细胞+核转液+空质粒37℃、5%CO2的培养箱中培养48h后查看细胞内荧光并收集细胞。ⅱ将micro RNA99和Lipid A分别作用于沉默后的巨噬细胞,实验分组如下:(1)实验组a:巨噬细胞-TLR8质粒+micro RNA99+转染试剂(2)对照组a:巨噬细胞-空质粒+micro RNA99+转染试剂(3)实验组b:巨噬细胞-TLR8质粒+LipidA(4)对照组b:巨噬细胞-空质粒+Lipid A37℃、5%CO2的培养箱中培养24h后收集上清液。(4)测量方法:(1)在倒置显微镜下观察THP-1细胞是否转化为巨噬细胞;(2)在荧光共聚焦下查看细胞内荧光判断质粒转染情况;(3)采用Western Blot方法测两组TLR8蛋白的表达水平;(4)用ELISA试剂盒测定细胞培养上清液中TNF-α的水平。结果:(1)THP-1向巨噬细胞转化的形态观察THP-1由圆形或类圆形,单个或葡萄串样聚集的悬浮状态转化为梭形或多边形的贴壁细胞,即巨噬细胞。(2)HIV派生的新型micro RNA99对巨噬细胞TNF-α释放的影响在micro RNA99实验组和Lipid A对照组,上清液中TNF-α的含量明显高于micro RNA-TAR对照组和空白对照组(p0.05),有统计学差异;micro RNA-TAR对照组和空白对照组无明显差异(p0.05),无统计学意义;micro RNA99实验组较Lipid A对照组,上清液TNF-α水平明显升高(p0.05),有统计学差异。(3)HIV派生的新型micro RNA99对巨噬细胞TNF-α释放的机制研究ⅰ在荧光共聚焦下查看细胞荧内光判断质粒转染情况巨噬细胞内呈现绿色荧光,说明TLR8质粒及对照质粒已转入巨噬细胞内。ⅱ采用Western Blot方法测两组TLR8蛋白的表达水平转染TLR8质粒细胞中TLR8蛋白表达量明显低于对照组(p0.05),有统计学差异。ⅲ用ELISA试剂盒测定上清液TNF-α的水平在micro RNA99实验组,上清液中TNF-α的含量明显低于micro RNA99对照组(p0.05),有统计学差异;Lipid A实验组与Lipid A对照组无明显差异(p0.05),无统计学意义。结论:1.micro RNA99促进巨噬细胞释放TNF-α;2.micro RNA99通过TLR8的途径影响巨噬细胞释放TNF-α。
[Abstract]:Aim: to investigate the effect of new micro RNA99 derived from HIV on the release of TNF- 伪 from macrophages and its mechanism. Methods: (1) the macrophage model was constructed and THP-1 (human acute monocytic leukemia cells) was stimulated by PMA to differentiate into macrophages. The morphological changes of macrophages were observed under inverted microscope. (2) the effect of new micro RNA99 derived from HIV on the release of TNF- 伪 from macrophages was affected by chemical synthetic substances. The groups were as follows: (1) Experimental group 1: macrophage transfer reagent micro RNA99 (2) control group 1: macrophage transfer reagent micro RNA-TRA (3) control group 2: macrophage Lipid A (4) control group 3: macrophage fine Cell medium 37 鈩，

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