【摘要】 目的观察RNA干扰细胞周期检测点激酶CHK1和CHK2表达对食管癌细胞照射后G2期阻滞的影响。方法CHK1和CHK2基因各选择4段序列分别设计合成短发卡状RNA (shRNA),分别与pENTRTM／U6质粒连接后转染Eca109食管癌细胞。采用Western blotting检测CHK1和CHK2蛋白表达,RT-PCR检测其mRNA表达,流式细胞仪检测5 Gy照射后细胞周期变化,克隆法检测5 Gy照射后细胞存活率。结果CHK1和CHK2基因各成功建立4个序列的shRNA连接质粒,转染Eca109细胞后均使其蛋白表达降低。用抑制效果最好的CHK1和CHK2 shRNA转染Eca109细胞后,其mRNA表达下降;在转染后72 h,子一代细胞CHK1和CHK2蛋白仍明显降低,并使5 Gy照射后1 h的CHK2-T68磷酸化水平降低,而对CHK1-S345磷酸化水平无明显影响。CHK1 shRNA转染的Eca109细胞在5 Gy照射后12 h时,明显减轻G2期阻滞程度;转染后72 h和5 Gy照射后12 h,子一代Eca109细胞G2期阻滞仍明显低于单纯5 Gy照射组;而CHK2 shRNA转染不影响照射后G2期阻滞。CHK1和CHK2 shRNA转染可降低5 Gy照射后Eca109细胞存活率。结论shRNA转染对CHK1和CHK2蛋白表达的抑制效应至少可以持续3 d,且可传递给子代细胞;CHK1 shRNA转染可以减轻Eca109细胞照射后G2期阻滞,增加放射敏感性。更多还原

【Abstract】 Objective To observe the effect of RNA interference on CHK1 and CHK2 expression and change of G2/M phase arrest in esophageal carcinoma cells after irradiation. Methods Four sequences short hairhip RNA (shRNA) of each CHK1 and CHK2 genes were constructed and connected with vector of pENTRTM/U6 plasmid, respectively, and then transfected into Eca109 cells with lipofectamine 2000 reagent. Protein and mRNA expression of CHK1 and CHK2 genes were detected with Western blotting and RT-PCR, respectively. Cell cycling was measured by flow cytometry after 5 Gy irradiation. Cell survival rate after 5 Gy irradiation was evaluated by clonegenetic assay. Results Four shRNA vector each of CHK1 and CHK2 genes were successfully constructed and transfected into Ecal09 cells, respectively. Protein expression of CHK1 and CHK2 were obviously decreased. Their mRNA expressions were also decreased after transfected with shRNA of CHK1 and CHK2. Arrest of G2/M stage in Ecal09 cells were obviously decreased only in cells transfected with CHK1 shRNA but not with CHK2 shRNA at 12 h after 5 Gy irradiation. In first progeny Eca109 cells transfected with CHK1 and CHK2 shRNA, expression of CHK1 and CHK2 protein was also decreased. The level of phosphorylated CHK2-T68 expression was decreased at 1 h after 5 Gy irradiation, and at 72 h only transfected with CHK2 shRNA but not with CHK1 shRNA. Phosphorylation level of CHK1-S345 was not increased after transfected with CHK1 or CHK2 shRNA, but arrest of G2/M stage still remained at 12 h after 5 Gy irradiation and at 72 h accordingly. The cell survival rate was decreased in Ecal09 cells transfected with CHK1 or CHK2 shRNA after 5 Gy irradiation. Conclusion After transfected with shRNA of CHK1 or CHK2, their expressions of mRNA and protein in Eca109 cells are markedly inhibited and this inhibition effect can be observed in their first progeny cells and at least hold for 3 days. Arrest of G2/M phase can be reduced after irradiation when teansfected with shRNA of CHK1 and the radiosensitivity of Ec109 cells can be increased.更多还原