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

【摘要】：目的:研究HIV派生的miR99能否引起巨噬細胞內(nèi)自噬及對自噬功能的影響,從而進一步對HIV慢性免疫激活的機制進行探討。方法:1.巨噬細胞獲得:人單核細胞白血病細胞(THP-1)在佛波酯(PMA)刺激48小時后分化為貼壁巨噬細胞,倒置光學顯微鏡下觀察細胞形態(tài)以鑒定。2.將巨噬細胞分為實驗組(miR99組)、雷帕霉素組(RP組)、空白對照組,每組處置分別為加miR99、加雷帕霉素、不加任何試劑。每組均設置重復組。3.在各組處置后的第2、4、8、12、24小時等時間點取各組細胞裂解提取蛋白;重復組于8小時時刮取細胞,離心制細胞團保存于3%戊二醛內(nèi),交校內(nèi)電鏡教研室制標本。4.透射電鏡下察看各組時間點自噬小體數(shù)并計數(shù);Western blot方法檢測自噬相關(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小時后懸浮的THP-1細胞絕大部分轉(zhuǎn)變?yōu)槎噙呅蔚�、貼壁巨噬細胞。2.透射電鏡下觀察,比之于空白對照組,RP組及miR99組自噬小體顯著增多(P0.05),RP組及miR99組自噬小體相比無顯著差異(P0.05)。3.目標蛋白的表達:(1)RP組:相較于空白對照組,LC3-Ⅱ的表達隨時間(2h、4h、8h、12h、24h)遞增(LC3-Ⅱ,P0.05);P62隨時間(2h、4h、8h、12h、24h)遞減(P62,P0.05)(2)mi R99組:相較于空白組,LC3Ⅱ的表達隨時間(2h、4h、8h、12h、24h)遞增(P0.05),P62于各時段幾乎無變化(P0.05)。(3)miR99組與RP組相比較,LC3-Ⅱ的表達均隨時間進展而遞增,但miR99組的P62不隨時間變化。結(jié)論:1.miR99引起巨噬細胞內(nèi)自噬發(fā)生。2.miR99抑制巨噬細胞內(nèi)自噬的降解功能。3.自噬體可能是巨噬細胞內(nèi)HIV的儲存庫之一。4.對自噬功能的影響可能參與HIV慢性免疫激活的機制。
[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：1979750