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目的:探讨干扰Wrap53基因表达对肿瘤细胞放射敏感性的作用及其分子机制。方法:构建针对Wrap53的干扰质粒;脂质体转染法转染至p53野生型(wtp53)的人骨肉瘤U2OS细胞;RT-PCR和蛋白质印迹法检测Wrap53和wtp53基因的mRNA和蛋白表达;克隆形成实验测定放射敏感性参数;流式细胞术(flow cytometry,FCM)分析细胞周期变化。结果:Wrap53的特异性shRNA质粒转染U2OS细胞后,细胞内Wrap53和wtp53的mRNA和蛋白表达水平均受到明显抑制;Wrap53基因干扰组U2OS细胞的D0值为0.44±0.01,明显高于阴性对照组0.37±0.01,P=0.001,Wrap53基因干扰组SF2值为0.72±0.10,高于阴性对照组0.44±0.02,P=0.047;Wrap53干扰细胞在放射后16h(P=0.014)和24h(P=0.011)的G2/M期阻滞更加明显,显著高于对照组;但Wrap53干扰细胞在放射后出现G1期阻滞减少,以放射后24h最为显著,P=0.001。结论:干扰Wrap53基因表达降低了U2OS细胞的放射线敏感性,该作用可能与下调wtp53表达和诱导细胞放射后G2/M期阻滞有关。
Objective: To investigate the effect of interfering with Wrap53 gene expression on tumor cell radiosensitivity and its molecular mechanism. Methods: The constructed shRNA targeting Wrap53 was transfected into U2OS cell line of human osteosarcoma with p53 wild type (wtp53) by lipofectamine. The mRNA and protein expression of Wrap53 and wtp53 were detected by RT-PCR and Western blotting. The radiosensitivity parameters were determined experimentally. The cell cycle was analyzed by flow cytometry (FCM). Results: The mRNA and protein expression levels of Wrap53 and wtp53 in Wrap53-shRNA transfected U2OS cells were all significantly inhibited. W053 cells in Wrap53-treated U2OS cells had a D0 value of 0.44 ± 0.01, which was significantly higher than that in the negative control group (P = 0.014) and 24h (P = 0.011, P = 0.011, P <0.01) .Wrap53 interference group SF2 value was 0.72 ± 0.10, higher than the negative control group 0.44 ± 0.02, P = 0.047; ) G2 / M phase arrest was more obvious, which was significantly higher than that of the control group; however, the arrest of G1 phase in Wrap53 interfering cells decreased after irradiation, and the most significant was 24h after irradiation (P = 0.001). Conclusion: Interfere with the expression of Wrap53 reduces the radiation sensitivity of U2OS cells, which may be related to the down-regulation of wtp53 expression and G2 / M arrest after induction of cell radiation.