發(fā)布時(shí)間：2019-05-14 04:11

【摘要】：目的:探討HIV派生的新型micro RNA99對(duì)巨噬細(xì)胞TNF-α釋放的影響及其機(jī)制。方法:(1)構(gòu)建巨噬細(xì)胞模型用PMA(佛波酯)刺激THP-1(人急性單核細(xì)胞白血病細(xì)胞)分化成巨噬細(xì)胞,于倒置顯微鏡下觀察細(xì)胞形態(tài)是否發(fā)生變化。(2)HIV派生的新型micro RNA99對(duì)巨噬細(xì)胞TNF-α釋放的影響以化學(xué)合成的物質(zhì)作用于巨噬細(xì)胞,分組如下:(1)實(shí)驗(yàn)組1:巨噬細(xì)胞+轉(zhuǎn)染試劑+micro RNA99(2)對(duì)照組1:巨噬細(xì)胞+轉(zhuǎn)染試劑+micro RNA-TRA(3)對(duì)照組2:巨噬細(xì)胞+Lipid A(4)對(duì)照組3:巨噬細(xì)胞+培養(yǎng)基37℃、5%CO2的培養(yǎng)箱中培養(yǎng)24小時(shí)收集并檢測(cè)上清液中TNF-α釋放的情況。(3)HIV派生的新型micro RNA99對(duì)巨噬細(xì)胞TNF-α釋放的機(jī)制研究ⅰTLR8質(zhì)粒轉(zhuǎn)染,實(shí)驗(yàn)分組如下:(1)實(shí)驗(yàn)組a:巨噬細(xì)胞+核轉(zhuǎn)液+TLR8質(zhì)粒(2)對(duì)照組b:巨噬細(xì)胞+核轉(zhuǎn)液+空質(zhì)粒37℃、5%CO2的培養(yǎng)箱中培養(yǎng)48h后查看細(xì)胞內(nèi)熒光并收集細(xì)胞。ⅱ將micro RNA99和Lipid A分別作用于沉默后的巨噬細(xì)胞,實(shí)驗(yàn)分組如下:(1)實(shí)驗(yàn)組a:巨噬細(xì)胞-TLR8質(zhì)粒+micro RNA99+轉(zhuǎn)染試劑(2)對(duì)照組a:巨噬細(xì)胞-空質(zhì)粒+micro RNA99+轉(zhuǎn)染試劑(3)實(shí)驗(yàn)組b:巨噬細(xì)胞-TLR8質(zhì)粒+LipidA(4)對(duì)照組b:巨噬細(xì)胞-空質(zhì)粒+Lipid A37℃、5%CO2的培養(yǎng)箱中培養(yǎng)24h后收集上清液。(4)測(cè)量方法:(1)在倒置顯微鏡下觀察THP-1細(xì)胞是否轉(zhuǎn)化為巨噬細(xì)胞;(2)在熒光共聚焦下查看細(xì)胞內(nèi)熒光判斷質(zhì)粒轉(zhuǎn)染情況;(3)采用Western Blot方法測(cè)兩組TLR8蛋白的表達(dá)水平;(4)用ELISA試劑盒測(cè)定細(xì)胞培養(yǎng)上清液中TNF-α的水平。結(jié)果:(1)THP-1向巨噬細(xì)胞轉(zhuǎn)化的形態(tài)觀察THP-1由圓形或類(lèi)圓形,單個(gè)或葡萄串樣聚集的懸浮狀態(tài)轉(zhuǎn)化為梭形或多邊形的貼壁細(xì)胞,即巨噬細(xì)胞。(2)HIV派生的新型micro RNA99對(duì)巨噬細(xì)胞TNF-α釋放的影響在micro RNA99實(shí)驗(yàn)組和Lipid A對(duì)照組,上清液中TNF-α的含量明顯高于micro RNA-TAR對(duì)照組和空白對(duì)照組(p0.05),有統(tǒng)計(jì)學(xué)差異;micro RNA-TAR對(duì)照組和空白對(duì)照組無(wú)明顯差異(p0.05),無(wú)統(tǒng)計(jì)學(xué)意義;micro RNA99實(shí)驗(yàn)組較Lipid A對(duì)照組,上清液TNF-α水平明顯升高(p0.05),有統(tǒng)計(jì)學(xué)差異。(3)HIV派生的新型micro RNA99對(duì)巨噬細(xì)胞TNF-α釋放的機(jī)制研究ⅰ在熒光共聚焦下查看細(xì)胞熒內(nèi)光判斷質(zhì)粒轉(zhuǎn)染情況巨噬細(xì)胞內(nèi)呈現(xiàn)綠色熒光,說(shuō)明TLR8質(zhì)粒及對(duì)照質(zhì)粒已轉(zhuǎn)入巨噬細(xì)胞內(nèi)。ⅱ采用Western Blot方法測(cè)兩組TLR8蛋白的表達(dá)水平轉(zhuǎn)染TLR8質(zhì)粒細(xì)胞中TLR8蛋白表達(dá)量明顯低于對(duì)照組(p0.05),有統(tǒng)計(jì)學(xué)差異。ⅲ用ELISA試劑盒測(cè)定上清液TNF-α的水平在micro RNA99實(shí)驗(yàn)組,上清液中TNF-α的含量明顯低于micro RNA99對(duì)照組(p0.05),有統(tǒng)計(jì)學(xué)差異;Lipid A實(shí)驗(yàn)組與Lipid A對(duì)照組無(wú)明顯差異(p0.05),無(wú)統(tǒng)計(jì)學(xué)意義。結(jié)論:1.micro RNA99促進(jìn)巨噬細(xì)胞釋放TNF-α;2.micro RNA99通過(guò)TLR8的途徑影響巨噬細(xì)胞釋放TNF-α。
[Abstract]:Aim: to investigate the effect of new micro RNA99 derived from HIV on the release of TNF- 偽 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- 偽 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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