针对小胶质细胞上BDNF表达的siRNA筛选及其抑制效应检测

Selection of the most effective small interfering RNA inhibiting the expression of BDNF in microglial cells and detection of its inhibitory effect

目的筛选有效抑制小胶质细胞上脑源性神经生长因子(brain-derived neurotrophic factor,BDNF)表达的小干扰RNA(small interference RNA,siRNA)序列,并观察其对小胶质细胞活性标记物OX-42表达的调节作用。方法设计并合成针对BDNF的siRNA3对(siRNA1588,siRNA283,siR-NA232)及一条带绿色荧光标记的通用阴性对照FAM-siRNA。在LipofectamineTM 2000介导下转染小胶质细胞。分别采用Real-TimePCR及Western blot方法测定并比较其抑制率;采用磺基罗丹明B法(sulforhodamine B,SRB)分析转染复合物的细胞毒性。同时采用免疫荧光组织化学结合共聚焦显微镜观察BDNF和OX-42的表达情况。结果①与阴性对照组及siRNA283、siRNA232相比,BDNFsiRNA1588(50μmol·L-1,脂质体与siRNA比例1∶3)干扰小胶质细胞后,其mR-NA和蛋白的相对表达量最低(P<0.01)。②Lipofectami-neTM 20008μl复合50μmol·L-1siRNA(脂质体与siRNA比例为1∶3),不仅细胞毒性低而且转染效率高(P<0.01),为最佳转染条件。③BNDF和OX-42存在共表达;且siR-NA1588作用后OX-42的平均光密度值(IOD/area)明显降低(P<0.01)。结论①siRNA1588对小胶质细胞上BDNF表达的抑制效果最大。②BDNF在小胶质细胞的活性调节中具有重要作用。

Aim To pick out the siRNA which could most effectively inhibit the expression of brain-derived neurotrophic factor（ BDNF） in microglial cells,to detect the cytotoxicity of the transfection complex,and to ob-serve the change of OX-42 expression,the microglial marker,after BDNF siRNA treatment. Methods Four siRNAs were chemically synthesized： three of them were used to inhibit BDNF expression in microglial cells,the rest was fluorescence-labeled mismatch siRNA as a negative control. They were all transfected into microglial cells,respectively. BDNF mRNA was detected 24 h after transfection by Real-Time PCR and itsprotein expression was observed done by Western blot 48 h later. The Sulforhodamine B（ SRB） assay was used to investigate the drug-induced cytotoxicity. Co-expres-sion pattern of BDNF and OX-42 was determined by double-labeling immunofluorescence. Results ① The BDNF siRNA1588 was the most effective siRNA,compared with the vehicle or mismatch siRNA-treated group（ P〈0. 01） . ② 8 μl LipofectamineTM 2000 and 50 μmol·L -1 siRNA（ 1 ∶ 3 ratio for liposome and siR-NA） had both lower cytotoxicity and higher transfection efficiency,which was suitable for transfection. ③ The coexistence of BDNF with OX-42 was demonstrated, and the expression of OX-42 significantly decreased compared with the control after siRNA1588 treatment. Conclusions ① The BDNF siRNA1588 can inhibit BDNF expression most effectively in microglial. ② BDNF plays an important role in the regulation of microglial activities.