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目的:研究辐射诱导表达载体pEgr-hPTEN体外转染对人脑胶质瘤SHG-44细胞周期进程及G1期激酶抑制蛋白P27表达的影响。方法:脂质体包裹重组载体体外转染肿瘤细胞并经G418筛选稳定表达细胞株,光学显微镜和透射电镜观察稳定转染细胞形态,以Western blotting法检测PTEN基因的辐射诱导表达情况,应用流式细胞术研究稳定转染联合0~10Gy X-射线照射对胶质瘤细胞周期进程及P27kip1的表达。结果:稳定转染PTEN基因的SHG-44细胞PTEN蛋白的相对表达量可被辐射诱导增强,5Gy以内PTEN蛋白的相对表达量随照射剂量增加而增加;稳定转染细胞超微结构有明显的退行性改变,可见核内染色质趋边的类似早期凋亡的改变;稳定转染联合X-射线照射细胞周期发生明显改变,细胞从G1期到S期发生阻滞,细胞周期相关蛋白P27表达上调。结论:PTEN基因稳定转染联合辐射可诱导肿瘤细胞G1期阻滞,并与P27kip1表达上调有关。
OBJECTIVE: To study the effect of radiation-induced expression vector pEgr-hPTEN on the cycle progression of human glioma SHG-44 cells and the expression of p27 kinase inhibitor G1 protein in vitro. METHODS: The recombinant plasmid was transfected into tumor cells in vitro and stably transfected into G418 cells. The morphology of cells was observed by light microscopy and transmission electron microscopy. The expression of PTEN gene was detected by Western blotting. The flow cytometry Cytometry Stable transfection combined with 0 ~ 10 Gy X-ray irradiation on the cell cycle progression and P27kip1 expression in glioma cells. Results: The relative expression of PTEN protein in SHG-44 cells stably transfected with PTEN gene was enhanced by radiation. The relative expression of PTEN protein in 5-Gy group increased with the increase of irradiation dose. The ultrastructure of stably transfected cells was obviously degenerated The results showed that the cell cycle changed from G1 phase to S phase, and the expression of cell cycle related protein P27 was up-regulated . CONCLUSION: Stable transfection of PTEN gene combined with radiation can induce G1 phase arrest in tumor cells, which is related to the up-regulation of P27kip1 expression.