Inhibition of Proliferation of Human Osteosarcoma Cells Transfected with PIN1 Antisense Gene

目的观察以pIRES2-EGFP质粒为载体的PIN1反义基因转染对人骨肉瘤细胞增殖的抑制作用。方法用不同量(0、20、50、100、200、250μL)的以pIRES2-EGFP质粒为载体包装的反义PIN1基因转染人骨肉瘤细胞系MG-63细胞,收集转染前后的培养细胞和上清液,用MTT法观测转染前后细胞生长曲线;用流式细胞仪观测细胞生长周期变化和细胞凋亡情况;用免疫印迹法(Western-blot)检测Pinl蛋白的表达变化;用逆转录聚合酶链式反应(RT-PCR)检测PIN1mRNA表达变化。结果MTT法和流式细胞仪显示反义PIN1基因转染能抑制细胞生长,促进其凋亡;Western-Blot结果表明转染反义Pin1基因的量越多,其灰度越低,测得空白对照组及不同量(0、20、50、100、200、250μL)的转染基因组其相应的吸光度比值分别为0．854±0．136、0．866±0．138、0．732±0．154、0．611±0．121、0．547±0．109、0．398±0．113、0．320±0．151,差异有显著性(P<0．05);RT-PCR检测其灰度比值分别为0．983±0．125、0．988±0．127、0．915±0．157、0．786±0．125、0．608±0．124、0．433±0．130、0．410±0．158,差异有显著件(P<0．05)。结论反义Pin1基因可封闭Pin1mRNA,使已转染反义Pin1基因的MG-63细胞表达的Pin1蛋白减少,从而抑制人骨肉瘤细胞MG- 63增殖。

Inhibition of proliferation of human osteosarcoma cells transfected with PIN1 antisense gene

Objective: To evaluate the inhibition of proliferation of human osteosarcoma cells transfected with Pin1 anti-sense gene. Methods: Different doses of antisense Pin1 gene (0, 20, 50, 100, 200, 250 μL) were transfected into osteosarcoma MG-63 cells. The cells and culture supernatant before and after transfection were collected. The curve of cell growth was made by MTT method. The cell growth cycle and apoptosis were detected by FCM. The expression of Pin1 was detected by Western-blot and that of Pin1 mRNA by polymerase chain reaction (RT-PCR) respectively. Results: MTT and FCM assays indicated that the transfection by antisense Pin1 gene could inhibit MG-63 proliferation and induce apoptosis. Western-blot assays revealed that the antisense Pin1 gene-transfected MG-63 cells had weaker staining than those without transfected with antisense Pin1 gene, and staining intensity was negatively related with doses. The cells transfected by different doses of gene (0, 20, 50, 100, 200, 250 μL) had different absorbance rate: 0.854±0.136, 0.866±0.138, 0.732±0.154, 0.611±0.121, 0.547±0.109, 0.398±0.113, 0.320±0.151 respectively, with the difference being significant by F and q test (P<0.05). The expression of Pin1 mRNA had the similar results and its absorbance rate was 0.983±0.125, 0.988±0.127, 0.915±0.157, 0.786±0.125, 0.608±0.124, 0.433±0.130, 0.410±0.158 respectively (P <0.05). Conclusion: The expression of Pin1 mRNA in MG-63 cells could be inhibited by antisense Pin1 gene, so to reduce the expression of Pin1 and depress the proliferation of human osteosarcoma cells MG-63.