本文選題：RNA干擾 + 樹(shù)突狀細(xì)胞　； 參考：《天津醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的：應(yīng)用RNAi(RNA interference)技術(shù)探討靶向MyD88的shRNA質(zhì)粒載體抑制大鼠骨髓源樹(shù)突狀細(xì)胞(Dendritic Cell, DC) MyD88基因表達(dá)的可行性。 方法：采用培養(yǎng)基選擇法經(jīng)體外培養(yǎng)大鼠骨髓源DC；流式細(xì)胞儀檢測(cè)DC表面抗原MHC-Ⅱ和OX-62表達(dá)情況；經(jīng)體外設(shè)計(jì)合成針對(duì)MyD88 mRNA序列特異性MyD88 shRNA3條,并分別構(gòu)建于Pgenesil.1質(zhì)粒,提取重組質(zhì)粒進(jìn)行酶切鑒定并測(cè)序；實(shí)驗(yàn)分為空白對(duì)照組、GenePorter 3000組、pHK-shRNA組、pMyD88 1-shRNA組、pMyD88 2-shRNA組和1pMyD88 3-shRNA組等六個(gè)組別。在陽(yáng)離子脂質(zhì)體轉(zhuǎn)染劑介導(dǎo)下轉(zhuǎn)染DC；熒光實(shí)時(shí)定量PCR檢測(cè)轉(zhuǎn)染后MyD88 mRNA表達(dá)水平的變化：WesternBlot技術(shù)檢測(cè)轉(zhuǎn)染后MyD88蛋白表達(dá)水平的變化。使用SPSS 17.0 for Windows進(jìn)行數(shù)據(jù)分析。 結(jié)果：經(jīng)體外培養(yǎng),每只大鼠可獲得1.0×107個(gè)DC；經(jīng)測(cè)序結(jié)果分析pMyD881-shRNA、pMyD882-shRNA、pMyD883-shRNA均為插入正確的克隆質(zhì)粒；熒光實(shí)時(shí)定量PCR檢測(cè)顯示構(gòu)建的3個(gè)pMyD88-shRNA質(zhì)粒通過(guò)脂質(zhì)體轉(zhuǎn)染大鼠骨髓源DC,均能不同程度地抑制MyD88的表達(dá),與對(duì)照組具有顯著差異(P0.01),其中pMyD88 1-shRNA質(zhì)粒組的抑制作用最為明顯,GenePorter 3000組與pHK-shRNA質(zhì)粒對(duì)照組間MyD88的mRNA轉(zhuǎn)錄水平無(wú)顯性差異(P0.05); WesternBlot檢測(cè)顯示3個(gè)MyD88-shRNA質(zhì)粒組中DC轉(zhuǎn)染后MyD88蛋白的的表達(dá)水平均較對(duì)照組明顯降低(P0.01),與對(duì)照組有顯著性差異,其中pMyD881-shRNA質(zhì)粒組抑制蛋白表達(dá)作用最為明顯,空白對(duì)照組、GenePorter3000組與pHK-shRNA質(zhì)粒對(duì)照組間MyD88蛋白表達(dá)水平無(wú)顯著性差異(P0.05)； 結(jié)論：經(jīng)體外培養(yǎng)可收獲大量大鼠骨髓源DC,形態(tài)典型。在細(xì)胞培養(yǎng)第12天,經(jīng)流式細(xì)胞儀檢測(cè),其MHC-Ⅱ和OX-62抗原表達(dá)分別為91.36%和72.70%。與空白對(duì)照組、GenePorter 3000組與pHK-shRNA質(zhì)粒對(duì)照組相比,pMyD88-shRNA質(zhì)粒組可高效、特異地抑制MyD88基因的mRNA和其蛋白的表達(dá),而其中pMyD881-shRNA質(zhì)粒抑制效率更高,可用于下一步實(shí)驗(yàn)研究。
[Abstract]:Aim: to investigate the feasibility of inhibiting the expression of MyD88 gene in rat bone marrow-derived dendritic cells by shRNA plasmid vector targeting MyD88 using RNAi(RNA interference technique. Methods: rat bone marrow derived DCs were cultured in vitro by medium selection method, the expression of DC surface antigen MHC- 鈪，

本文編號(hào)：1797003