论文部分内容阅读
目的:利用针对B7-H1的siRNA在脑胶质瘤U87细胞中下调B7-H1的表达,研究B7-H1的表达下调对U87细胞增殖和凋亡的影响。方法:根据人B7-H1的mRNA序列设计并合成两对针对B7-H1的siRNA,于转染前一天将U87细胞接种于10 cm细胞培养皿中,以转染时细胞融合度为50%-80%为宜,分别用250μl OPTI-MEM稀释15μl siRNA与15μl转染试剂Lipofectamine 2000,将两者混匀室温静置20 min后,滴入细胞培养皿中。转染60或72 h后利用流式细胞术检测B7-H1在细胞表面的表达情况,利用荧光定量PCR和Western Blot分别检测B7-H1的mRNA和蛋白质水平,并用MTT和流式细胞术检测B7-H1表达下调后,U87细胞增殖和凋亡的改变。结果:与阴性对照组相比,两对针对B7-H1的siRNA均可有效下调U87细胞中B7-H1的表达,流式细胞术结果显示与阴性对照组相比,B7-H1阳性表达率由53.2%分别下调至26.7%和20.6%;MTT结果显示B7-H1表达下调后U87细胞的增殖被显著抑制,培养第5天吸光度值由0.58±0.02分别下降至0.451±0.02及0.342±0.016;流式细胞术结果显示B7-H1表达下调后U87细胞凋亡明显,早期凋亡率由0.1%分别增加至12.8%及13.2%。结论:B7-H1的表达下调可有效抑制脑胶质瘤U87细胞的增殖,并促进细胞凋亡,提示B7-H1可能作为脑胶质瘤基因治疗的一个潜在靶点。
OBJECTIVE: To study the effect of down-regulation of B7-H1 expression on the proliferation and apoptosis of U87 cells by siRNA targeting B7-H1 downregulated B7-H1 expression in glioma U87 cells. METHODS: Two pairs of siRNA targeting B7-H1 were designed and synthesized based on the human B7-H1 mRNA sequence. U87 cells were seeded into 10 cm cell culture dishes on the day before transfection. The cell fusion degree was 50% 80% is appropriate, respectively, with 250μl OPTI-MEM diluted 15μl siRNA and 15μl transfection reagent Lipofectamine 2000, the two were mixed at room temperature for 20 minutes, then dropped into the cell culture dish. The expression of B7-H1 on the cell surface was detected by flow cytometry at 60 or 72 hours after transfection. The mRNA and protein levels of B7-H1 were detected by real-time PCR and Western Blot. The expression of B7-H1 was detected by MTT and flow cytometry -H1 expression down-regulated U87 cell proliferation and apoptosis changes. Results: Compared with the negative control group, two siRNAs against B7-H1 effectively down-regulated the expression of B7-H1 in U87 cells. The results of flow cytometry showed that the positive rate of B7-H1 53.2% were down-regulated to 26.7% and 20.6%, respectively. The MTT results showed that the proliferation of U87 cells was significantly inhibited after B7-H1 was down-regulated, and the absorbance decreased from 0.58 ± 0.02 to 0.451 ± 0.02 and 0.342 ± 0.016 Cytometry showed that apoptosis of U87 cells was significantly decreased after B7-H1 expression was down-regulated, and early apoptosis rate increased from 0.1% to 12.8% and 13.2% respectively. Conclusion: The down-regulation of B7-H1 can effectively inhibit the proliferation and promote the apoptosis of glioma U87 cells, suggesting that B7-H1 may serve as a potential target for gene therapy of glioma.