摘要
为实现对肺癌A549细胞的EMT示踪,构建了E-cadherin基因启动子的慢病毒质粒,并与包装质粒pVSVG、Δ8.91共转染HEK293T细胞,包装成有活性的慢病毒,收取病毒感染A549细胞,利用流式细胞仪和Western blot检测感染成功.进一步用TGF-β诱导感染成功的A549细胞,通过在荧光显微镜下观察荧光变化、荧光定量PCR和Western blot检测GFP和E-cadherin的变化,发现在TGF-β诱导48 h后,A549细胞形态由鹅卵石样变成长梭形.并且荧光显微镜观察到相比较于未诱导的细胞,诱导后的A549细胞绿色荧光明显变暗,同时GFP和E-cadherin的RNA水平和蛋白质表达水平均降低.以E-cadherin基因启动子为基础建立了A549细胞的EMT荧光示踪体系,为进一步研究肺癌的EMT过程提供了材料.
In order to trace EMT status of lung cancer A549 cells,we constructed a lentiviral plasmid carrying E-cadherin gene promoter,which was co-transfected with packaging plasmids pVSVG andΔ8.91 into HEK 293T cells to produce active lentivirus.Then the A549 cells were infected by the collected lentivirus,and the success of infection was examined by flow-cytometry and Western blot.Afterwards,TGF-βwas used to treat the infected A549 cell,and the expression of GFP and E-cadherin in those cells was detected by the fluorescence observation,RT-PCR and Western blot.We found that after 48 hours of TGF-βinduction,the morphology of A549 cells changed from cobblestone to long fusiform.Fluorescence microscopy showed that fluorescence signal of A549 cells was significantly decreased in the induction group compared with untreated cells,and RNA levels and protein levels of GFP and E-cadherin were decreased as well.This indicated that the EMT tracer system was established successfully.In conclusion,we established a EMT tracer system using lentivirus in the A549 cells,providing materials for further study of EMT process of lung cancer.
作者
谭拥军
吴东丽
余雳
陈燕
TAN Yongjun;WU Dongli;YU Li;CHEN Yan(College of Biology,Hunan University,Changsha 410082,China)
出处
《湖南大学学报（自然科学版）》
EI
CAS
CSCD
北大核心
2020年第12期131-136,共6页
Journal of Hunan University:Natural Sciences
基金
国家自然科学基金资助项目(84172718)。
