PTEN基因稳定转染联合辐射对肿瘤细胞周期进程及G1期激酶抑制蛋白P27kip1表达的影响

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P27^kip1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P27^kip1表达上调有关。

Effects of PTEN transfer on cell cycle progression and expression of P27kip1 followed by X-ray irradiation

Objective To investigate the effects of pEgr-hPTEN stable transfer combined with irradiation on the cell cycle progression and the expression of cell cycle kinase inhibitor P27kip1 protein of SHG-44 human glioma cells.Methods pEgr-hPTEN vector containing the exogenous wild type PTEN gene was transfected into SHG-44 cells under mediation of lipofectamine in vitro,the positive cell clones were selected and amplified by using G418.Western blotting was used to measure the expression of PTEN protein.Transmission electron microscope was adopted to detect the cell ultrastructural changes and flow cytometry was adopted to analysis the changes of cell cycle progression and the expression of P27kip1 in SHG-44-sPTEN cells followed by different doses of X-ray irradiation.Results Egr-1 promoter could be induced and activated by irradiation and then enhanced the expression of downstream PTEN gene within 5Gy.The ultrastructure of SHG-44-sPTEN cells had many degenerative changes and many early apoptotic changes including the chromosome condensate around the nuclear envelope.pEgr-hPTEN stable transfer combined with X-ray irradiation could significantly induce G1 arrest.The expression of P27kip1 proteins increased in SHG-44-sPTEN stable transfected cells.Conclusion PTEN stable transfer combined with irradiation can significantly induce G1 arrest.The molecular basis may be correlated with the enhanced expression of PTEN induced by irradiation and increased expression of cell cycle kinase inhibitor P27kip1.