【摘要】 背景与目的:肿瘤细胞照射后常表现为细胞周期的变化,本研究采用细胞周期监测点激酶CHK1和CHK2基因的短发卡状RNA(shRNA)转染食管癌细胞,观察对其蛋白表达以及60Coγ射线照射后细胞周期的影响。方法:设计合成并构建质粒连接的CHK1和CHK2 shRNA,分别提取质粒DNA并采用脂质体转染TE13细胞并传代;采用Westernblot、RT-PCR和流式细胞仪分别检测CHK1和CHK2蛋白和mRNA表达、以及5Gy60Coγ射线照射后细胞周期变化,克隆形成实验检测5Gyγ射线照射后细胞存活率。结果:采用CHK1和CHK2shRNA转染TE13细胞后,其mRNA和蛋白表达均明显降低。单纯5Gyγ射线照射后24h,TE13细胞G2/M期比例由未照射组的32.17%增至61.47%;shRNA转染的TE13细胞在5Gyγ射线照射后24h,G2/M期比例由单纯照射组的61.47%降至28.13%(CHK1)和42.80%(CHK2),P<0.05。5Gyγ射线、CHK1shRNA加5Gyγ射线、以及CHK2shRNA加5Gyγ射线照射后,细胞存活率分别为27.0%、13.0%和21.0%。shRNA转染的子一代TE13细胞在转染后120h,对CHK1和CHK2蛋白表达的抑制作用已基本消失;转染120h后以5Gyγ射线照射24h,CHK1或CHK2 shRNA转染组G2/M期比例高于单纯照射组,P<0.05;至转染后144h和5Gyγ射线照射后48h,转染组与单纯照射组比较G2/M期比例无明显差别,P>0.05。结论:采用质粒连接的人CHK1和CHK2 shRNA转染TE13细胞后,可以明显抑制其mRNA和蛋白表达并消除照射后G2/M期阻滞,增加放射敏感性;提示TE13细胞γ射线照射后G2/M期检测点可能受CHK1和CHK2激酶双重调节,但以CHK1为主。更多还原

【Abstract】 BACKGROUND & OBJECTIVE: Cell cycle of tumor cells often changes after irradiation. This study was to observe the effect of RNA interference (RNAi) on mRNA and protein expression of human checkpoint kinases CHK1 and CHK2 and on cell cycle of esophageal carcinoma cell line TE13 after irradiation. METHODS: Short hairpin RNAs (shRNAs) of CHK1 and CHK2 genes were designed and constructed, and then transfected into TE13 cells. The expression of CHK1 and CHK2 were detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Cell cycle was measured by flow cytometry after 5-Gy radiation. Cell survival after irradiation was evaluated with colony forming assay. RESULTS: After transfection of CHK1 and CHK2 shRNAs, the mRNA and protein expression of CHK1 and CHK2 in TE13 cells were inhibited obviously. At 24 h after 5-Gy radiation, G2/M phase proportion was higher in irradiated TE13 cells than in control TE13 cells (61.47% vs. 32.17%), and significantly lower in CHK1 shRNA- and CHK2 shRNA-transfected TE13 cells than in untransfected TE13 cells (28.13% and 42.80% vs. 61.47%, P<0.05); cell survival rates were 27% in simplex irradiation group, 14% in CHK1 shRNA transfection plus irradiation group, and 21% in CHK2 shRNA transfection plus irradiation group. In the first generation of transfected TE13 cells, the inhibition of CHK1 and CHK2 expression was almost diminished at 120 h after transfection. At 120 h after transfection and 24 h after 5-Gy irradiation, G2/M phase proportion was significantly higher in CHK1 shRNA- and CHK2 shRNA-transfected groups than in simplex irradiation group (P<0.05); but at 144 h after transfection and 48 h after 5-Gy irradiation, the difference between transfection groups and simplex irradiation group was not significant (P>0.05). CONCLUSIONS: RNAi could inhibit the expression of CHK1 and CHK2, release G2/M phase arrest after irradiation, and enhance radiosensitivity of TE-13 cells. G2/M phase of TE-13 cells after irradiation may be regulated by CHK1 and CHK2.更多还原