CAJ | 학술논문

目的探讨ErbB-2转录因子1 (TOB1)对人类肺癌A549细胞放射敏感性的影响并探讨其机制.方法通过脂质体介导的重组质粒转染及G418筛选,建立TOB1高表达的肺癌A549细胞稳定转染株;采用克隆形成实验检测TOB1对A549细胞放射敏感性的影响;采用单细胞凝胶电泳实验检测TOB1对电离辐射引起的DNA损伤修复的影响;同时采用Western blot实验检测TOB1对DNA损伤修复相关蛋白表达的调控作用.结果通过重组质粒转染及G418筛选,成功建立TOB1的肺癌A549细胞稳定转染株;克隆形成实验证实外源性TOB1高表达显著增加肺癌A549细胞的放射敏感性,采用单击多靶模型拟合细胞存活曲线,细胞的放射增敏比(SER)为1.63;单细胞凝胶电泳实验证实接受6Gy的X线照射后,A549/TOB1细胞的尾矩分别为(25.71±4.32)％,明显高于A549细胞[(14.26±1.21)％,P＜0.05].Western blot实验证实与A549母细胞比较,X线照射后A549/TOB1细胞中共济失调毛细血管扩张症突变基因(ATM)、06-甲基鸟嘌呤-DNA甲基转移酶(MGMT)、DNA依赖性蛋白激酶催化亚单位(DNA-PKcs)、Ku70、Ku80、DNA双链断裂修复基因X线修复交叉互补4(XRCC4)蛋白的表达水平明显降低.结论 TOB1高表达增加肺癌A549细胞的放射敏感性,其机制可能与TOB1抑制DNA损伤修复相关蛋白的表达,从而抑制DNA损伤的修复有关.
목적 탐토ErbB-2전록인자1 (TOB1)대인류폐암A549세포방사민감성적영향병탐토기궤제.방법 통과지질체개도적중조질립전염급G418사선,건립TOB1고표체적폐암A549세포은정전염주;채용극륭형성실험검측TOB1대A549세포방사민감성적영향;채용단세포응효전영실험검측TOB1대전리복사인기적DNA손상수복적영향;동시채용Western blot실험검측TOB1대DNA손상수복상관단백표체적조공작용.결과 통과중조질립전염급G418사선,성공건립TOB1적폐암A549세포은정전염주;극륭형성실험증실외원성TOB1고표체현저증가폐암A549세포적방사민감성,채용단격다파모형의합세포존활곡선,세포적방사증민비(SER)위1.63;단세포응효전영실험증실접수6Gy적X선조사후,A549/TOB1세포적미구분별위(25.71±4.32)％,명현고우A549세포[(14.26±1.21)％,P＜0.05].Western blot실험증실여A549모세포비교,X선조사후A549/TOB1세포중공제실조모세혈관확장증돌변기인(ATM)、06-갑기조표령-DNA갑기전이매(MGMT)、DNA의뢰성단백격매최화아단위(DNA-PKcs)、Ku70、Ku80、DNA쌍련단렬수복기인X선수복교차호보4(XRCC4)단백적표체수평명현강저.결론 TOB1고표체증가폐암A549세포적방사민감성,기궤제가능여TOB1억제DNA손상수복상관단백적표체,종이억제DNA손상적수복유관.
Objective To evaluate the effect of transducer of ErbB2.1 (TOB1) on radiosensitivity of human lung cancer cell line A549 and investigate the related mechanisms.Methods The lipofectamine mediated TOB1 recombinant plasmid transfection and selective G418 cell culture were performed to obtain lung cancer cell line A549 with over-expressed TOB1.The effects on lung cancer cell radiosensitivity in vitro were evaluated by clonogenic survival analysis,and comet DNA damage assay was used to understand the molecular mechanisms of radiosensitization of TOB1 and investigate its effects on the initial DNA damage response to irradiation.Western blotting analysis was performed to determine the related mechanisms.Results Using Lipofectamine and G418-mediated plasmid stable transfection,multiple clones stably transfected with TOB1 were selected.As compared with parental and mock transfected cells,enhanced radiosensitivity was detected in A549/TOB1 cells and the sensitizing enhancement ratio was 1.63.Comet DNA damage assay revealed that the tail moment of A549/TOB1 cells were (25.71 ± 4.32) ％,which was significantly higher than that of A549 cells.Western blotting analysis revealed that TOB1 could down-regulate the expression of ATM,MGMT,DNA-PKcs,Ku70,Ku80 and XRCC4 as compared with parental cells.Conclusion Overexpression of TOB1 enhanced the radiosensitivity of A549 cells in vitro,at least by its potential function as an expression regulator of DNA damage repair related proteins,as well as its regulation on DNA damage repair.