Construction of Cdc42 Expression Vector and the Effect of It Coordinating with the Peptidomimetics on Cell Migration

Abstract

为了研究Cdc42协同拟肽化合物对A549细胞迁移的影响，构建携带红、绿荧光报告基因的Cdc42真核表达载体，转染293T细胞，观察基因表达情况；测定实验用化合物(PMC4)对A549细胞的毒性；进一步将Cdc42转染高迁移率的A549细胞，然后用拟肽化合物刺激，通过细胞划痕实验，初步检测Cdc42协同拟肽化合物对A549细胞迁移的影响。结果表明：本实验成功构建了Cdc42异构体基因的真核表达载体pGFP-cdc42-2、pGFP-cdc42-3、pRed-cdc42-2、pRed-cdc42-3，转染293T细胞效率约为90%；10 μmol/L的拟肽化合物对A549细胞毒性微弱，拟肽药物分子末端对位上的取代基为氟时，其对细胞迁移具有促进作用，过表达Cdc42时，Cdc42协同药物使细胞迁移进一步增加，具体机制有待进一步研究。 In order to study the effect of Cdc42 coordinating with the peptidomimetics on the migration of A549 cells, the Cdc42 eukaryotic expression vectors harboring fluorescent reporter genes were constructed and transfected into 293T cells. The expression of Cdc42 was observed by fluorescence microscopy. The cytotoxicity of the compound (PMC4) on A549 cells was tested. The Cdc42 genes were further ex- pressed in the high mobility cells A549 and the compound was added. The cooperation effect of Cdc42 with the compound on the cell migration was investigated by cell wound scratch assay. The results showed that the eukaryotic expression vectors of Cdc42 isoform genes were successfully constructed named pGFP-cdc42-2, pGFP-cdc42-3, pRed-cdc42-2, pRed-cdc42-3 respectively. These recombinants plasmids were transfected into 293T cells and the transfection ef-ficiency was about 90%. 10 μmol/L peptidomimetic compounds showed weak toxicity to A549 cells. When the substituent group at the terminal para-position of the peptidomimetic drug molecule is F (fluorin), it can increase the migration of A549 cells. While the Cdc42 gene was over-expressed in A549 cells and the cell migration rate was enhanced furtherly. More studies and results need to be carried out to reveal the detail mechanism.