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背景与目的:利用小干扰RNA(smallinterferingRNA,siRNA)抑制哺乳动物基因表达已成为研究基因功能的一种有效方法。本研究采用siRNA抑制人神经胶质瘤细胞系U-87细胞上容积调控氯通道ClC-2基因的表达,观察其受抑制后对细胞增殖能力的影响。方法:设计和构建两个针对人ClC-2基因的siRNA真核表达载体,并给予酶切鉴定和DNA序列分析鉴定;用脂质体LipofectamineTM2000介导转染,将空载体质粒pSUPER.puro-shRNA和两个重组质粒pSUPER.puro-shRNA-ClC-21、pSUPER.puro-shRNA-ClC-22分别转染入U-87细胞(依次为PP0组、PP1组和PP2组);采用RT-PCR检测ClC-2mRNA表达变化;MTT分析检测细胞活性;流式细胞仪检测细胞周期;平板克隆形成实验检测克隆形成率。结果:目的片段成功地连接到真核细胞表达载体pSUPER.puro上。PP1组、PP2组与对照组、PP0组相比较,ClC-2基因的mRNA水平明显降低,细胞生长速度明显减慢,细胞周期进程被阻滞在G1期:细胞的G1期百分含量分别增加了约30.24%、18.04%(P<0.05)。平板克隆形成试验发现,克隆形成率PP1组[(11.0±1.0)%]、PP2组[(20±3.1)%]明显低于对照组[(46.5±1.6)%]和PP0组[(47.5±2.8)%](P<0.01)。结论:干扰人神经胶质瘤细胞系U-87细胞的ClC-2基因表达可以抑制细胞的增殖,提示ClC-2基因可能成为控制人恶性胶质瘤生长的新靶点。
BACKGROUND & OBJECTIVE: Suppression of mammalian gene expression by small interfering RNA (siRNA) has become an effective method for studying gene function. In this study, siRNA was used to inhibit the ClC-2 gene expression in the volume-regulated chloride channel of human glioma cell line U-87, and its effect on cell proliferation was observed. Methods: Two eukaryotic expression vectors targeting human ClC-2 gene were designed and constructed. The recombinant plasmid was identified by restriction enzyme digestion and DNA sequence analysis. LipofectamineTM2000 was used to transfect the empty plasmid pSUPER.puro-shRNA And two recombinant plasmids pSUPER.puro-shRNA-ClC-21 and pSUPER.puro-shRNA-ClC-22 were transfected into U-87 cells (PP0 group, PP1 group and PP2 group, respectively) ClC-2mRNA expression changes; MTT assay to detect cell activity; flow cytometry to detect cell cycle; plate clone formation assay to detect the rate of clone formation. Results: The target fragment was successfully ligated into the eukaryotic cell expression vector pSUPER.puro. Compared with control group and PP0 group, the mRNA level of ClC-2 gene in PP1 group and PP2 group was significantly decreased, the cell growth rate was significantly slowed down, and the cell cycle progression was arrested in G1 phase: the percentage of cells in G1 phase increased About 30.24%, 18.04% (P <0.05). The results of plate clone formation assay showed that the clonogenic rates in PP1 group [(11.0 ± 1.0)%] and PP2 group [(20 ± 3.1)%] were significantly lower than those in control group [(46.5 ± 1.6)%] and 2.8%] (P <0.01). Conclusion: The expression of ClC-2 gene in human glioma cell line U-87 can inhibit the proliferation of human glioma cell line U-87, suggesting that ClC-2 gene may be a new target for the control of human malignant glioma.