脂质体RNAiMAX介导siRNA转染沉默星形胶质细胞AQP4基因

目的明确脂质体RNAiMAX(lipofectamine RNAiMAX)是否可以介导小干扰RNA(siRNA)转染入原代培养星形胶质细胞并实现水通道蛋白4(AQP4)基因沉默。方法利用原代培养的Wistar大鼠大脑皮层星形胶质细胞,通过倒置荧光显微镜和Tecan酶标仪,观察lipofectamine RNAiMAX是否能介导Cy3标记的siRNA转染入细胞内,以及RNAiMAX浓度和siRNA浓度对转染效率的影响;利用Real-time PCR方法检测AQP4 siRNA转染的基因沉默效果。结果倒置荧光显微镜和Tecan酶标仪检测发现,随着siRNA和RNAiMAX浓度的增加,转染细胞荧光强度随之增加(n=6);Real-time PCR检测结果显示,3ml/L RNAiMAX和30nmol/L AQP4 siRNA以及6ml/L RNAiMAX和60nmol/L AQP4 siRNA转染星形胶质细胞24h、48h、72h时,AQP4 mRNA水平均降低80%以上(n=6)。结论 Lipofectamine RNAiMAX可以介导siRNA转染入原代培养星形胶质细胞中并实现AQP4基因的沉默,3ml/L RNAiMAX和30nmol/L AQP4 siRNA即可达到理想的沉默效果。

Silencing AQP4 gene in the astrocytes through siRNA transfection by lipofectamine RNAiMAX

Objective To determine whether siRNA transfection with the help of Lipofectamine RNAiMAX can achieve AQP4 gene silencing in primary cultured astrocytes. Methods Primary cultured astrocytes from Wistar rat’s cerebral cortex were used in this study. Fluorescence microscope and Tecan microplate reader were used to examine whether Cy3-labeled siRNA can be transfected into astrocytes by lipofectamine RNAiMAX and the effects of different RNAiMAX concentration and siRNA concentration on the transfection efficiency. The silencing effect of AQP4 gene was detected by Real-time PCR. Results There were a lot of transfected cells under different transfection conditions as observed by fluoscence microscope. Tecan micropalte reader detection showed that the fluorescence intensity enhanced when siRNA and RNAiMAX concentrations increased. The level of AQP4 mRNA decreased more than 80 percent at 24 hours, 48 hours and 72 hours after 3ml/L RNAiMAX and 30nmol/L AQP4 siRNA or 6ml/L RNAiMAX and 60nmol/L AQP4 siRNA were used to transfection observed by Real-time PCR(n=6). Conclusion The fact that AQP4 mRNA significantly decreased after transfection indicates that siRNA can be successfully transfected into primary cultured astrocytes and silenced AQP4 gene by using Lipofectamine RNAiMAX.