CAJ | 학술논문

目的构建针对cortactin基因干扰的胃癌SGC-7901稳定细胞株.方法设计3条针对皮质肌动蛋白(cortactin)基因的短发卡RNA(shRNA)序列以及l条对照序列,退火后插入到PLKO.1慢病毒质粒载体中,质粒构建完成后和辅助质粒psPAX2,PCMV-VSVG共转染HEK293T细胞,包装成慢病毒颗粒,感染胃癌SGC-7901细胞株,并使用嘌呤霉素筛选.最终筛选出3个细胞株,提取该3株细胞的RNA和蛋白质分别进行实时定量聚合酶链反应(Real-time PCR)和Western blot检测cortactin基因的mRNA和蛋白的表达.结果在3株不同的稳定细胞株中,PLKO.1+shRNA3细胞株cortactin基因的mRNA和蛋白质表达下降最为明显,约为对照组的10％.结论成功构建出针对cortactin基因干扰的胃癌SGC-7901稳定细胞株.
목적 구건침대cortactin기인간우적위암SGC-7901은정세포주.방법 설계3조침대피질기동단백(cortactin)기인적단발잡RNA(shRNA)서렬이급l조대조서렬,퇴화후삽입도PLKO.1만병독질립재체중,질립구건완성후화보조질립psPAX2,PCMV-VSVG공전염HEK293T세포,포장성만병독과립,감염위암SGC-7901세포주,병사용표령매소사선.최종사선출3개세포주,제취해3주세포적RNA화단백질분별진행실시정량취합매련반응(Real-time PCR)화Western blot검측cortactin기인적mRNA화단백적표체.결과 재3주불동적은정세포주중,PLKO.1+shRNA3세포주cortactin기인적mRNA화단백질표체하강최위명현,약위대조조적10％.결론 성공구건출침대cortactin기인간우적위암SGC-7901은정세포주.
Objective To establish a stable SGC-7901 gastric cancer cell line with cortactin downregulation.Methods Three pairs of primers specific to knockdown cortactin and a pair of control primers were designed and synthesized.The primers were annealed and inserted into PLKO.1 plasmids.The plasmids,psPAX2,and PCMV-VSVG were transfected into HEK293T cells simultaneously,and then lentiviral particles were produced and collected.The lentiviral particles were used to infect SGC-7901 cells and puromycin was used to select stable cell lines.Three stable cell lines were selected at last.Real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting were conducted to detect the expression level of cortactin mRNA and protein respectively.Results The expression levels of cortactin mRNA and protein were significantly down-regulated.The mRNA and protein levels of cortactin in PLKO.1 + short hairpin RNA (shRNA)3 cell line were significantly decreased,about 10％ of the control stable cell line.Conclusion The stable SGC-7901 gastric cell lines with cortactin down-regulation were established successfully.