中华脑科疾病与康复杂志（电子版）
中華腦科疾病與康複雜誌（電子版）
중화뇌과질병여강복잡지（전자판）
CHINESE JOURNAL OF BRAIN DI8SEASES AND REHABILITATIN(ELECTRONIC EDITION)
2014年
6期
45-48
,共4页
杨碧莹%彭晴霞%潘经锐%王艺东
楊碧瑩%彭晴霞%潘經銳%王藝東
양벽형%팽청하%반경예%왕예동
小神经胶质细胞%RNA,小分子干扰%Toll样受体9
小神經膠質細胞%RNA,小分子榦擾%Toll樣受體9
소신경효질세포%RNA,소분자간우%Toll양수체9
Microglia%RNA,small interfering%Toll like receptor 9
目的：筛选有效抑制小鼠小胶质细胞（ BV-2细胞）上Toll样受体9（ TLR9）表达的小干扰RNA（ siRNA）序列，并检测转染复合物的细胞毒性。方法设计并合成针对TLR9的3对siRNA （siRNA-836、siRNA-1549和siRNA-2410）及一条带绿色荧光标记（FAM）的通用阴性对照FAM-siRNA。在X-tremeGENE siRNA转染试剂介导下转染BV-2细胞。倒置荧光显微镜下观察转染后BV-2细胞FAM-siRNA的分布，流式细胞仪检测转染效率， q-PCR检测TLR9的mRNA表达， Western blot 检测TLR9蛋白的表达，并比较其抑制率，筛选出有效抑制靶基因表达的siRNA。 CCK-8检测细胞存活率的变化。结果 BV-2细胞转染FAM-siRNA后6 h，胞质内可见绿色荧光分布。 FAM-siRNA表达阳性率为（91.87±4.77）％。转染TLR9 siRNA后，BV-2细胞中TLR9 mRNA表达明显下降。与阴性对照组及序列 siRNA-836、siRNA-2410相比，序列 siRNA-1549的 TLR9沉默效能最高，使 BV-2细胞上TLR9 mRNA表达于24 h和48 h分别降低（77.89±4.23）％和（65.36±11.07）％， TLR9蛋白表达分别下降（48.37±20.56）％和（44.30±14.31）％。 CCK-8检测TLR9 siRNA转染组与对照组相应时间点比较，BV-2细胞存活率无明显变化（ P＞0.05）。结论序列siRNA-1549对BV-2细胞上TLR9表达的抑制效果最大，利用RNA干扰处理BV-2细胞，对细胞基本无毒性作用。
目的：篩選有效抑製小鼠小膠質細胞（ BV-2細胞）上Toll樣受體9（ TLR9）錶達的小榦擾RNA（ siRNA）序列，併檢測轉染複閤物的細胞毒性。方法設計併閤成針對TLR9的3對siRNA （siRNA-836、siRNA-1549和siRNA-2410）及一條帶綠色熒光標記（FAM）的通用陰性對照FAM-siRNA。在X-tremeGENE siRNA轉染試劑介導下轉染BV-2細胞。倒置熒光顯微鏡下觀察轉染後BV-2細胞FAM-siRNA的分佈，流式細胞儀檢測轉染效率， q-PCR檢測TLR9的mRNA錶達， Western blot 檢測TLR9蛋白的錶達，併比較其抑製率，篩選齣有效抑製靶基因錶達的siRNA。 CCK-8檢測細胞存活率的變化。結果 BV-2細胞轉染FAM-siRNA後6 h，胞質內可見綠色熒光分佈。 FAM-siRNA錶達暘性率為（91.87±4.77）％。轉染TLR9 siRNA後，BV-2細胞中TLR9 mRNA錶達明顯下降。與陰性對照組及序列 siRNA-836、siRNA-2410相比，序列 siRNA-1549的 TLR9沉默效能最高，使 BV-2細胞上TLR9 mRNA錶達于24 h和48 h分彆降低（77.89±4.23）％和（65.36±11.07）％， TLR9蛋白錶達分彆下降（48.37±20.56）％和（44.30±14.31）％。 CCK-8檢測TLR9 siRNA轉染組與對照組相應時間點比較，BV-2細胞存活率無明顯變化（ P＞0.05）。結論序列siRNA-1549對BV-2細胞上TLR9錶達的抑製效果最大，利用RNA榦擾處理BV-2細胞，對細胞基本無毒性作用。
목적：사선유효억제소서소효질세포（ BV-2세포）상Toll양수체9（ TLR9）표체적소간우RNA（ siRNA）서렬，병검측전염복합물적세포독성。방법설계병합성침대TLR9적3대siRNA （siRNA-836、siRNA-1549화siRNA-2410）급일조대록색형광표기（FAM）적통용음성대조FAM-siRNA。재X-tremeGENE siRNA전염시제개도하전염BV-2세포。도치형광현미경하관찰전염후BV-2세포FAM-siRNA적분포，류식세포의검측전염효솔， q-PCR검측TLR9적mRNA표체， Western blot 검측TLR9단백적표체，병비교기억제솔，사선출유효억제파기인표체적siRNA。 CCK-8검측세포존활솔적변화。결과 BV-2세포전염FAM-siRNA후6 h，포질내가견록색형광분포。 FAM-siRNA표체양성솔위（91.87±4.77）％。전염TLR9 siRNA후，BV-2세포중TLR9 mRNA표체명현하강。여음성대조조급서렬 siRNA-836、siRNA-2410상비，서렬 siRNA-1549적 TLR9침묵효능최고，사 BV-2세포상TLR9 mRNA표체우24 h화48 h분별강저（77.89±4.23）％화（65.36±11.07）％， TLR9단백표체분별하강（48.37±20.56）％화（44.30±14.31）％。 CCK-8검측TLR9 siRNA전염조여대조조상응시간점비교，BV-2세포존활솔무명현변화（ P＞0.05）。결론서렬siRNA-1549대BV-2세포상TLR9표체적억제효과최대，이용RNA간우처리BV-2세포，대세포기본무독성작용。
Objective To pick out the small interfering RNA ( siRNA) which could inhibit Toll like receptor9(TLR9) in mouse microglial cells most effectively and detect the cytotoxicity of the transfection complex.Methods Four siRNAs were chemically synthesized:three of them were used to inhibit TLR 9 expression in microglial cells , the rest was fluorescencelabeled mismatch siRNA as a negative control .They were all transfected into microglial cells by using X-tremeGENE siRNA transfection reagent , respectively . Transfection rates were measured by flow cytometry .Q-PCR and western blot were used to detect TLR 9 expression at 24 h and 48 h.CCK-8 assays were carried out to determine the changes of cell viabilities after TLR9 gene silencing .Results FAM-siRNA fluorescence had diffuse cytoplasmic distribution with little nuclear stain at 6 h after the transfection under fluorescent microscopy .Transfection efficiency was (91.87 ± 4.77 )%detected by flow cytometry .Among the three siRNAs ,TLR9 mRNA levels decreased most obviously in siRNA-1549 which decreased (77.89 ±4.23 )%and(65.36 ±11.07 )%at 24 h and 48 h.TLR9 protein levels in siRNA-1549 decreased (48.37 ±20.56 )%and (44.30 ±14.31 )%at 24 h and 48 h.Cell survival rate did not change significantly when TLR 9 siRNA was transfected into BV-2 cells compared to control group ( P>0.05 ) .Conclusion siRNA-1549 can inhibit TLR9 expression most effectively in BV-2 microglial cells.RNAi has low cytotoxicity,which is suitable for transfecting BV-2 microglial cells.
