PDX1真核表达载体的构建及其对PANC-1细胞PI3K/AKT信号途径的影响

Construction of eukaryotic expression vector of PDX1 gene and the effect of PDX1 on PI3K/AKT pathway in PANC-1

目的构建胰腺十二指肠同源框蛋白1(PDX1)基因真核表达载体并转染至PANC-1细胞,观察其对PI3K/AKT信号通路的影响。方法从Hela细胞中提取总RNA,逆转后PCR扩增PDX1编码区片段,然后将PDX1的CDS插入PCMV6-Entry质粒构建PCMV6-Entry-PDX1表达重组体。重组体转染至PANC-1细胞中通过G418筛选出稳定表达细胞株。Western blot分析PDX1蛋白水平表达及p-AKT水平变化。结果 PDX1基因成功在PANC-1中表达。利用Western blot检测验证获得了稳定的PDX1过表达细胞株。Western blot结果显示:过表达PDX1的细胞株AKT表达含量与正常细胞及对照细胞没有区别而p-AKT水平降低。结论PDX1基因过表达能够降低PANC-1细胞的p-AKT水平。

Objective To construct eukaryotic expression vetor of PDX1 gene, and study the effect of PDX1 on PI3K/AKT pathway in PANC-1. Methods Total RNA was isolated from Hela cell, followed by synthesis of com- plementary DNA（cDNA） and CDS of PDX1 gene was obtained by PCR. The expression vetor of PCMV6-Entry- PDX1 was constructed by inserting the CDS of PDX1 into PCMV6-Entry plasmid, and then PANC-1 cell transfected with PCMV6-Entry- PDX1 was screened by G418 to get stable overexpression cells. Western blot was performed to detect the level of PDX1, p-AKT and AKT. Results PDX1 was expressed in PANC-1 cells successfully. The Western blot results showed that the expression level of AKT had no difference between PDX1 overexpession cells and control cells and vector cells, while the level of p-AKT decreased in PDX1 overexpression cells, compared with the control cells. Conclusion Overexpression PDX1 can decrease the level of p-AKT in PANC-1 cells.