磷酸酶基因PTEN对骨肉瘤细胞凋亡机制研究

Research of PTEN Gene on Apoptosis in Osteosarcoma Cells

目的探讨肿瘤抑制基因PTEN对人骨肉瘤细胞系MG-63细胞凋亡的影响及其分子作用机制。方法将携带有野生型PTEN及绿色荧光蛋白的腺病毒(Ad-PTEN-GFP)及空载体腺病毒(Ad-GFP),转染MG-63细胞系,流式细胞仪分析转染效率及细胞凋亡率,半定量RT-PCR检测PTEN mRNA水平变化,Western blot法检测PTEN蛋白表达,试剂盒检测caspase-3、caspase-7、caspase-9蛋白活性。结果以感染复数为100,转染MG-63细胞2天后腺病毒感染效率即达93.2%±4.7%。转染PTEN基因5天后,细胞凋亡率为29.8%,细胞形态出现明显凋亡改变。分子学检测结果显示转染PTEN基因后,PTEN mRNA及蛋白表达明显升高;caspase-3、caspase-7、caspase-9蛋白活性明显增加。结论 PTEN基因可能通过提高caspase-3、caspase-7、caspase-9活性,诱导人骨肉瘤MG-63细胞凋亡。

Objective To investigate the mechanism of tumor - suppressing gene PTEN on the cell apoptosis and the influence of Caspase family on human osteosarcoma cell line MG -63 cells in vitro. Methods The Reeomhinated adenovirus which contained PTEN gene（Ad- PTEN- GFP） and the empty vector （Ad -GFP） were transfected into MG- 63 cells respectively. The transfection efficiency of Ad - PTEN - GFP and apoptosis rate were assessed by flow cytometry （ FCM）. The apoptosis was detected by morphological character- istics of light microscope. The PTEN mRNA levels were detected by reverse transcriptional PCR. Western blot was used to detected PTEN protein. Caspase- 3,caspase- 7 and caspase- 9 protein activities were tested by kits. Results When multiplicity of infection （MOI） was 100, the efficiency of denovirns transfection MG - 63 cell was 93.2% ＋ 4.7% after transfection 48h. The apoptosis ratio was 29.8% after MG -63 cells transfected with Ad - PTEN - GFP 5 days. The apoptosis appearance with morphological characteristics appeared, PTEN mRNA and protein increased, the caspase -3, caspase -7, caspase -9 protein activity was up -regulated respectively compared with Ad - GFP group after transfaction of PTEN gene. Conclusion PTEN gene can induce MG - 63 ceils apoptosis probability via up - regulating the caspase - 3, caspase - 7, caspase - 9 ability.