Establishment and Validation of a Fluorescent Tracer System Based on E-cadherin Promoter
Author:
Affiliation:
Fund Project:
摘要
|
图/表
|
访问统计
|
参考文献
|
相似文献
|
引证文献
|
资源附件
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.
Abstract:
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 and Δ8.91 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-β was 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-β induction，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.
