siMDR1基因转染人类BT325胶质瘤细胞系的表达

目的探讨siMDR1基因转染人类胶质母细胞瘤细胞BT325后细胞的表达能力。方法设计并合成siRNA质粒,取培养对数期生长良好的胶质母细胞系BT325,传代培养。将阳离子脂质体(Lipofectamine 2000)和质粒MDR1 DNA按比例共转染。瞬时对转染成功的细胞系应用抗性药物-嘌呤霉素进行筛选,筛出稳定的细胞系后,分别在荧光显微镜下分析转染情况。通过免疫细胞化学染色及图像分析对瞬时转染和稳定转染的BT325细胞进行检测,确定MDR1基因产物P-糖蛋白(P-gp)表达水平。阿霉素对瞬时转染及稳定转染的BT325耐药性进行分析。结果成功构建了逆转录病毒si RNA质粒载体,经过瞬时转染BT325细胞生长状态良好,48h表达绿色荧光最强。加入嘌呤霉素筛选后,在第8天出现单细胞克隆。免疫细胞化学染色证实瞬时转染与稳定转染BT325细胞P-gp的表达下降。细胞的转染效率为70%～80%。BT325细胞经过RNA干扰,细胞对MDR1耐药性明显下降,IC_(50)降低,细胞的耐药因子(RF)有所升高。结论siMDR1基因瞬时转染和稳定转染都可以抑制P-gp蛋白的表达,其可以作为基因治疗的重要手段。

Gene expression of human glioblastoma cell line BT325 transfected with siMDR1 gene in vitro

Objective To explore the ability to express siMDR1 gene in human glioblastoma cell line BT325 in vitro. Methods Design and construct siRNA plasmid vector. The cultured BT325 glioblastoma cell was transfected with Lipofectamine 2000 and MDR1 DNA plasmid mixture. The transient expression of BT325 cell was detected by immunocytochemistry to estimate P-glycoprotein expression after 48h transfection. And the screened drug puromycin was used to acquire stable expression BT325 cell. In addition it was measured too. Muhidrug resistance was analysis by DOX. Results siRNA plasmid vectors were constructed successfully and were confirmed by electrophoresis. After transient transfection the BT325 glioma cells grew well and the single cell clone which grew stably was successfully screened by the plasmid resistance drug 8 days after transfection. Immunocytochemistry demonstrated that P-gp expression reduced in both transient expression and stable expression groups. The transfection rate was 70% - 80%. The results of chemosensitivity assays showed that the transfected cells could increase the sensitivity of Dox and the survival rate of transfected cells decreased definitly. Conclusions Transient expression and stable expression of si MDR1 in BT325 cell could inhibit P-gp expression. And it will become an important method for the gene therapy of glioblastoma.