中华临床医师杂志（电子版）
中華臨床醫師雜誌（電子版）
중화림상의사잡지（전자판）
CHINESE JOURNAL OF CLINICIANS(ELECTRONIC VERSION)
2013年
22期
10140-10143
,共4页
薛卓维%熊苗%蒋荣珍%李黎%滕银成
薛卓維%熊苗%蔣榮珍%李黎%滕銀成
설탁유%웅묘%장영진%리려%등은성
RNA,小分子干扰%树突细胞%慢病毒载体%C型凝集素受体
RNA,小分子榦擾%樹突細胞%慢病毒載體%C型凝集素受體
RNA,소분자간우%수돌세포%만병독재체%C형응집소수체
RNA,small interfering%Dendritic cells%Lentiviral vector%C-type lectin receptor
目的：构建C型凝集素受体（C-type lectin receptor，CLRs）过表达/siRNA慢病毒载体，并将其瞬时转染树突状细胞（dendritic cell，DC）。方法免疫磁珠分离、培养及鉴定胎盘和蜕膜来源的DC；构建CLRs过表达/siRNA慢病毒载体并转染DC；抽提转染CLRs过表达/siRNA慢病毒后的DC的总RNA；RT-PCR检测DC转染慢病毒前后CLRs基因mRNA表达量。结果从胎盘分别纯化得到的DC经流式细胞术检测DC纯度为（82.6±2.4）%；根据Genebank数据库成功构建了过表达CLRs基因慢病毒及CLRs基因siRNA慢病毒，病毒量为1×1010 Unit；将构建的两种慢病毒转染DC后，在荧光倒置显微镜下可观察到被转染的阳性细胞有绿色荧光蛋白（GTP）表达，通过流式细胞仪检测，转染效率分别为85%及82%左右；RT-PCR检测发现过表达CLRs基因慢病毒转染DC后mRNA相对含量为（14.26±0.47）%，较空白未转染的DC表达量[（1.67±0.19）%]明显上升（P＜0.01）；CLRs基因siRNA慢病毒转染DC后CLRs mRNA相对含量为（0.03±0.01）%，较空白未转染的 DC 表达量明显下降（P＜0.01）。结论成功构建了表达人CLRs-siRNA的慢病毒载体，它能有效沉默CLRs基因在胎盘DC中的表达并获得了其瞬时转染的DC；成功构建了过表达CLRs的慢病毒载体，它能有效地在体内发挥基因过表达效应并获得了其瞬时转染的DC。
目的：構建C型凝集素受體（C-type lectin receptor，CLRs）過錶達/siRNA慢病毒載體，併將其瞬時轉染樹突狀細胞（dendritic cell，DC）。方法免疫磁珠分離、培養及鑒定胎盤和蛻膜來源的DC；構建CLRs過錶達/siRNA慢病毒載體併轉染DC；抽提轉染CLRs過錶達/siRNA慢病毒後的DC的總RNA；RT-PCR檢測DC轉染慢病毒前後CLRs基因mRNA錶達量。結果從胎盤分彆純化得到的DC經流式細胞術檢測DC純度為（82.6±2.4）%；根據Genebank數據庫成功構建瞭過錶達CLRs基因慢病毒及CLRs基因siRNA慢病毒，病毒量為1×1010 Unit；將構建的兩種慢病毒轉染DC後，在熒光倒置顯微鏡下可觀察到被轉染的暘性細胞有綠色熒光蛋白（GTP）錶達，通過流式細胞儀檢測，轉染效率分彆為85%及82%左右；RT-PCR檢測髮現過錶達CLRs基因慢病毒轉染DC後mRNA相對含量為（14.26±0.47）%，較空白未轉染的DC錶達量[（1.67±0.19）%]明顯上升（P＜0.01）；CLRs基因siRNA慢病毒轉染DC後CLRs mRNA相對含量為（0.03±0.01）%，較空白未轉染的 DC 錶達量明顯下降（P＜0.01）。結論成功構建瞭錶達人CLRs-siRNA的慢病毒載體，它能有效沉默CLRs基因在胎盤DC中的錶達併穫得瞭其瞬時轉染的DC；成功構建瞭過錶達CLRs的慢病毒載體，它能有效地在體內髮揮基因過錶達效應併穫得瞭其瞬時轉染的DC。
목적：구건C형응집소수체（C-type lectin receptor，CLRs）과표체/siRNA만병독재체，병장기순시전염수돌상세포（dendritic cell，DC）。방법면역자주분리、배양급감정태반화세막래원적DC；구건CLRs과표체/siRNA만병독재체병전염DC；추제전염CLRs과표체/siRNA만병독후적DC적총RNA；RT-PCR검측DC전염만병독전후CLRs기인mRNA표체량。결과종태반분별순화득도적DC경류식세포술검측DC순도위（82.6±2.4）%；근거Genebank수거고성공구건료과표체CLRs기인만병독급CLRs기인siRNA만병독，병독량위1×1010 Unit；장구건적량충만병독전염DC후，재형광도치현미경하가관찰도피전염적양성세포유록색형광단백（GTP）표체，통과류식세포의검측，전염효솔분별위85%급82%좌우；RT-PCR검측발현과표체CLRs기인만병독전염DC후mRNA상대함량위（14.26±0.47）%，교공백미전염적DC표체량[（1.67±0.19）%]명현상승（P＜0.01）；CLRs기인siRNA만병독전염DC후CLRs mRNA상대함량위（0.03±0.01）%，교공백미전염적 DC 표체량명현하강（P＜0.01）。결론성공구건료표체인CLRs-siRNA적만병독재체，타능유효침묵CLRs기인재태반DC중적표체병획득료기순시전염적DC；성공구건료과표체CLRs적만병독재체，타능유효지재체내발휘기인과표체효응병획득료기순시전염적DC。
Objective To construct CLRs gene overexpressed/CLRs siRNA lentiviral vector and use them to transiently transfect DC. Methods Immunomagnetic separation and purification of DC from decidua and placenta of normal pregnant women; To Construct CLRs gene overexpressed/siRNA lentiviral vector, then use them to transfect DC; Extraction of total RNA from DC transfected by CLRs overexpressed/siRNA lentivirus;The mRNA expression levels of CLRs gene were detected by RT-PCR in DC transfected by CLRs gene overexpressed lentiviral vector or CLRs siRNA lentiviral vector. Results The purity of DC isolated from placenta was(82.6 ± 2.4)% which detected by FCM; We successfully constructed CLRs gene overexpressed/siRNA lentiviral vector, and the amount of virus(viral load) is 1×1010 Unit;Green fluorescence was observed in DC by means of an inverted fluorescence microscope and the transfection rate were respectively about 85%and 82%after transfected by CLRs gene overexpressed lentiviral vector and CLRs siRNA lentiviral vector. The CLRs mRNA in DC transfected by CLRs gene overexpressed lentiviral(14.26±0.47)%were higher than untransfected DC(1.67± 0.19)%which were dected by RT-PCR(P<0.01);in addition, the CLRs mRNA in DC transfected by CLRs siRNA lentiviral vector(0.03±0.01)% were obviously lower than untransfected DC(P<0.01). Conclusion The CLRs siRNA lentiviral vector was constructed successfully and it can effectively silence the CLRsmRNA expression in DC;The CLRs gene overexpressed lentiviral vector was constructed successfully and it can effectively express the CLRs mRNA in DC.
