有效抑制小鼠小胶质细胞上Toll样受体9表达的siRNA的筛选

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-tremeGENEsiRNA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，Westernblot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6h，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TLR9siRNA后，BV-2细胞中TLR9mRNA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TLR9mRNA表达于24h和48h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TLR9siRNA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。

Selection of an effective small interfering RNA inhibiting the expression of Toll like receptor 9 in microglial cells

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.