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目的 :探讨同源异型盒D10(homeobox D10,HOXD10)基因在人胆管癌细胞放疗增敏中的作用及其可能的作用机制。方法:用不同剂量的放射线照射胆管癌RBE和HCCC9810细胞后,应用实时荧光定量PCR法检测细胞中HOXD10 mRNA的表达。应用克隆形成实验、“多靶单击模型”和FCM法检测HOXD10稳定过表达的胆管癌LvHOXD10-RBE和Lv-HOXD10-HCCC9810细胞的放射增敏效应及细胞的凋亡情况,蛋白质印迹法检测放射治疗对HOXD10过表达的胆管癌LvHOXD10-RBE和Lv-HOXD10-HCCC9810细胞中凋亡相关蛋白表达的影响。结果:2、4和8 Gy放射治疗后48和72 h,胆管癌RBE和HCCC9810细胞中HOXD10 mRNA的表达水平均显著高于放疗后24 h组(P值均<0.05)。在2、4和8 Gy放射治疗后,Lv-HOXD10-RBE和LvHOXD10-HCCC9810细胞的克隆形成率均分别显著低于对应的仅表达绿色荧光蛋白的阴性对照Lv-CTRL-RBE和Lv-CTRL-HCCC9810细胞(P值均<0.05);Lv-HOXD10-RBE和Lv-HOXD10-HCCC9810细胞的平均致死剂量(mean lethal dose,D0)、准阈剂量(quasithreshold dose,Dq)和2 Gy单次照射后细胞的存活分数(survival fraction with 2 Gy,SF2)值均分别低于Lv-CTRL-RBE和Lv-CTRL-HCCC9810细胞(P值均<0.05)。4 Gy放射治疗后24 h,Lv-HOXD10-RBE和Lv-HOXD10-HCCC9810细胞凋亡率高于Lv-CTRL-RBE和Lv-CTRL-HCCC9810细胞(P值均<0.05);Lv-HOXD10-RBE和Lv-HOXD10-HCCC9810细胞中p53、p21和Bax蛋白的表达水平均高于Lv-CTRL-RBE和Lv-CTRLHCCC9810细胞,而双微体2(murine double minute 2,Mdm2)蛋白的表达水平均低于Lv-CTRL-RBE和Lv-CTRL-HCCC9810细胞(P值均<0.05)。结论:HOXD10可增加人胆管癌RBE和HCCC9810细胞对放射的敏感性,这一作用可能与凋亡相关蛋白的表达上调有关。
Objective: To investigate the role of homeobox D10 (HOXD10) gene in radiosensitization of human cholangiocarcinoma cells and its possible mechanism. Methods: After the RBE and HCCC9810 cells were irradiated with different doses of radiation, the expression of HOXD10 mRNA was detected by real-time fluorescence quantitative PCR. The radiosensitizing effect and cell apoptosis of LvHOXD10-RBE and Lv-HOXD10-HCCC9810 cells stably overexpressed by HOXD10 were detected by the method of clonogenic assay, the “multi-target click model” and the FCM method. Western blotting The effects of radiotherapy on the expression of apoptosis-related proteins in LvHOXD10-RBE and Lv-HOXD10-HCCC9810 cells overexpressing HOXD10 were studied. Results: The expressions of HOXD10 mRNA in RBE and HCCC9810 cells of 48 h and 72 h after radiotherapy were significantly higher than those of 24 h after radiotherapy (P <0.05). The clonogenic rates of Lv-HOXD10-RBE and LvHOXD10-HCCC9810 cells were significantly lower than those of the corresponding negative control Lv-CTRL-RBE and Lv-CTRL- The mean lethal dose (D0), quasithreshold dose (Dq) of Lv-HOXD10-RBE and Lv-HOXD10-HCCC9810 cells in HCCC9810 cells (P <0.05) The cell survival fraction with 2 Gy (SF2) values were lower than those of Lv-CTRL-RBE and Lv-CTRL-HCCC9810 cells respectively (all P <0.05). The apoptosis rates of Lv-HOXD10-RBE and Lv-HOXD10-HCCC9810 cells after 4 Gy radiation therapy were higher than those of Lv-CTRL-RBE and Lv-CTRL-HCCC9810 cells (all P values <0.05) The expression levels of p53, p21 and Bax in Lv-HOXD10-HCCC9810 cells were higher than those in Lv-CTRL-RBE and Lv-CTRLHCCC9810 cells, while the expression levels of murine double minute 2 (Mdm2) In Lv-CTRL-RBE and Lv-CTRL-HCCC9810 cells (P <0.05). CONCLUSION: HOXD10 can increase the radiosensitivity of human cholangiocarcinoma RBE and HCCC9810 cells, which may be related to the up-regulation of apoptosis-related proteins.