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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。
Objective To determine whether lipofectamine RNAiMAX could mediate the transfection of small interfering RNA (siRNA) into primary cultured astrocytes and to achieve AQP4 gene silencing. Methods Wistar rat primary cortical astrocytes were cultured and transfected with Cy3-labeled siRNA by Lipofectamine RNAiMAX by inverted fluorescence microscope and Tecan microplate reader. The RNAiMAX concentration and siRNA concentration On the transfection efficiency; Real-time PCR method was used to detect AQP4 siRNA transfection gene silencing effect. Results Fluorescence microscope and Tecan microplate reader showed that the fluorescence intensity of transfected cells increased with the increase of siRNA and RNAiMAX concentration (n = 6). Real-time PCR results showed that 3ml / L RNAiMAX and 30nmol / L AQP4 siRNA and 6ml / L RNAiMAX and 60nmol / L AQP4 siRNA transfected astrocytes 24h, 48h, 72h, AQP4 mRNA levels were reduced by more than 80% (n = 6). Conclusion Lipofectamine RNAiMAX can mediate siRNA transfection into primary cultured astrocytes and achieve silencing of AQP4 gene. 3ml / L RNAiMAX and 30nmol / L AQP4 siRNA can achieve the desired silencing effect.