本文關(guān)鍵詞：RNAi抑制小鼠樹突細(xì)胞共刺激通路的體外研究　出處：《天津醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

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【摘要】： 目的:本課題應(yīng)用RNAi技術(shù)修飾小鼠骨髓來(lái)源樹突細(xì)胞(Dendritic Cell,DC),敲減DC表面共刺激分子B7-1(CD80)及B7-2(CD86)表達(dá),探討RNAi對(duì)DC表面抗原CD80、CD86表達(dá)的影響以及誘導(dǎo)T淋巴細(xì)胞無(wú)能的機(jī)理。 方法:采用培養(yǎng)基細(xì)胞因子選擇法體外培養(yǎng)小鼠骨髓來(lái)源DC,利用已構(gòu)建的針對(duì)B7-1(CD80)及B7-2(CD86)的shRNA質(zhì)粒載體pB7shRNA轉(zhuǎn)染DC,流式細(xì)胞儀檢測(cè)轉(zhuǎn)染前后DC表面抗原CD80、CD86、MHCⅡ、CD11c表達(dá)情況,混合淋巴細(xì)胞培養(yǎng)觀察pB7shRNA質(zhì)粒轉(zhuǎn)染DC前后對(duì)激活異系T淋巴細(xì)胞增殖能力的影響,熒光實(shí)時(shí)定量PCR測(cè)定轉(zhuǎn)染前后CD80、CD86mRNA及混合淋巴細(xì)胞培養(yǎng)體系IL-2mRNA表達(dá)水平。使用SPSS 15.0軟件包進(jìn)行數(shù)據(jù)分析。 結(jié)果:經(jīng)過(guò)體外培養(yǎng),每只小鼠可獲得1.5-2×10~7個(gè)骨髓來(lái)源DC,細(xì)胞具備典型樹突狀結(jié)構(gòu),pB7shRNA質(zhì)粒載體轉(zhuǎn)染DC后經(jīng)流式細(xì)胞儀檢測(cè)其表面抗原CD80、CD86表達(dá)變化由92.812±1.377%、70.510±1.947%分別下降至31.137±1.702%、40.511±1.555%,與對(duì)照質(zhì)粒轉(zhuǎn)染組相比有顯著差異(P＝0.000)。而MHCⅡ、CD11c表達(dá)無(wú)明顯變化。熒光實(shí)時(shí)定量PCR檢測(cè)pB7shRNA質(zhì)粒轉(zhuǎn)染小鼠骨髓源DC后,CD80、CD86mRNA表達(dá)水平下降至33.481±2.823%、37.431±2.218%,與對(duì)照質(zhì)粒轉(zhuǎn)染組相比具有顯著差異(P＝0.000)�；旌狭馨图�(xì)胞培養(yǎng)顯示,pB7shRNA干擾DC對(duì)異系T淋巴細(xì)胞的刺激指數(shù)下降,與對(duì)照質(zhì)粒轉(zhuǎn)染組相比有顯著差異(P＝0.000),反應(yīng)體系中IL-2mRNA表達(dá)水平下降至25.675±1.521%,與對(duì)照質(zhì)粒轉(zhuǎn)染組相比具有顯著差異(P＝0.000)。 結(jié)論:培養(yǎng)基細(xì)胞因子選擇法可收獲大量的骨髓源DC。脂質(zhì)體介導(dǎo)pB7shRNA質(zhì)粒載體轉(zhuǎn)染小鼠骨髓DC可高效、特異地抑制B7-1及B7-2分子的表達(dá),使DC激活異系T淋巴細(xì)胞能力下降,混合淋巴細(xì)胞反應(yīng)體系中IL-2分泌減少,誘導(dǎo)T細(xì)胞無(wú)能。
[Abstract]:Objective: to modify mouse bone marrow-derived dendritic cells with RNAi technique. The expression of costimulatory molecules B7-1 (CD80) and B7-2 (CD86) on DC surface was reduced to investigate the effect of RNAi on DC surface antigen (CD80). The effect of CD86 expression and the mechanism of inducing T lymphocyte anergy. Methods: DC derived from mouse bone marrow was cultured in vitro by medium cytokine selection method. The constructed shRNA plasmid pB7shRNA for B7-1mCD80 and B7-2mCD86) was used to transfect DC. Flow cytometry was used to detect the expression of CD80, CD86, MHC 鈪，

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