本文選題：miR99 + 巨噬細(xì)胞��； 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:研究HIV派生的miR99能否引起巨噬細(xì)胞內(nèi)自噬及對(duì)自噬功能的影響,從而進(jìn)一步對(duì)HIV慢性免疫激活的機(jī)制進(jìn)行探討。方法:1.巨噬細(xì)胞獲得:人單核細(xì)胞白血病細(xì)胞(THP-1)在佛波酯(PMA)刺激48小時(shí)后分化為貼壁巨噬細(xì)胞,倒置光學(xué)顯微鏡下觀察細(xì)胞形態(tài)以鑒定。2.將巨噬細(xì)胞分為實(shí)驗(yàn)組(miR99組)、雷帕霉素組(RP組)、空白對(duì)照組,每組處置分別為加miR99、加雷帕霉素、不加任何試劑。每組均設(shè)置重復(fù)組。3.在各組處置后的第2、4、8、12、24小時(shí)等時(shí)間點(diǎn)取各組細(xì)胞裂解提取蛋白;重復(fù)組于8小時(shí)時(shí)刮取細(xì)胞,離心制細(xì)胞團(tuán)保存于3%戊二醛內(nèi),交校內(nèi)電鏡教研室制標(biāo)本。4.透射電鏡下察看各組時(shí)間點(diǎn)自噬小體數(shù)并計(jì)數(shù);Western blot方法檢測(cè)自噬相關(guān)蛋白P62及LC3Ⅱ,Image J軟件行半定量分析。5.收集資料,分析比較各組結(jié)果。結(jié)果:1.PMA刺激48小時(shí)后懸浮的THP-1細(xì)胞絕大部分轉(zhuǎn)變?yōu)槎噙呅蔚�、貼壁巨噬細(xì)胞。2.透射電鏡下觀察,比之于空白對(duì)照組,RP組及miR99組自噬小體顯著增多(P0.05),RP組及miR99組自噬小體相比無(wú)顯著差異(P0.05)。3.目標(biāo)蛋白的表達(dá):(1)RP組:相較于空白對(duì)照組,LC3-Ⅱ的表達(dá)隨時(shí)間(2h、4h、8h、12h、24h)遞增(LC3-Ⅱ,P0.05);P62隨時(shí)間(2h、4h、8h、12h、24h)遞減(P62,P0.05)(2)mi R99組:相較于空白組,LC3Ⅱ的表達(dá)隨時(shí)間(2h、4h、8h、12h、24h)遞增(P0.05),P62于各時(shí)段幾乎無(wú)變化(P0.05)。(3)miR99組與RP組相比較,LC3-Ⅱ的表達(dá)均隨時(shí)間進(jìn)展而遞增,但miR99組的P62不隨時(shí)間變化。結(jié)論:1.miR99引起巨噬細(xì)胞內(nèi)自噬發(fā)生。2.miR99抑制巨噬細(xì)胞內(nèi)自噬的降解功能。3.自噬體可能是巨噬細(xì)胞內(nèi)HIV的儲(chǔ)存庫(kù)之一。4.對(duì)自噬功能的影響可能參與HIV慢性免疫激活的機(jī)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，

本文編號(hào)：1979750