Differential Expression Of GV143-EGFP-ECAD And PIRES2-EGFP-ECAD Plasmids In HMrSV5Cells

Objective:Using pIRES2-EGFP-ECAD and GV143-EGFP-ECAD transfect into peritoneal mesothelium cells separately, and Evaluate the transfection efficiency, Then Investigate the overexpression of EC AD on HMrSV5cells.Methods:(1) By double enzyme digestion, recombination, the pla-smid of the ECAD was successfully constructed. After that, the plasmid of ECAD combined with pIRES2-EGFP or GV143-EGFP transfected into the Escherichia coli DH5alpha cells. Then positive recombinants were selected in the amplified cells. By amplification, extraction, enzyme identification and sequence analysis, the recombinants were constructed.(2) The two different recombinants were Transfected to HMrSV5cells by cationic liposome transfection method. The HMrSV5cells were divided into5groups according to the time of transfection:Oh,24h,36h,48and72h group.(3) EGFP(enhanced green fluorescent protein) positive cells were counted under the fluorescence microscope to calculate the transfection efficiency.(4) The total RNA were extracted after transfection, the ECAD mRNA expression level were detected by Real-Time PCR, Western blot and indirect immunofluorescence were used to detect the protein expression of ECAD in each groups, the protein expression of ECAD and a-SMA of empty plasmid transfection group on different time points were also detected.Results:(1)No mutation plasmid sequences of pIRES2-EGFP-ECAD and GV143-EGFP-ECAD plasmids were identified by DNA sequencing. It reveals that the enzyme-digested product of pIRES2-EGFP-ECAD located on2.6K and5.4K and GV143-EGFP-ECAD located on2.6K and4.8K. And it proves that plasmids is successful constructed.(2) There only a few of GV143-EGFP-ECAD transfected cells expresses EGFP (Enhanced green fluorescent protein) in36h and48h, in24h and72h have no EGFP expression. While the transfection efficiency of pIRES2-EGFP-ECAD transfected cells on24h、36h、48h、72h are18%、58%、41%、15%by separately.(3) The results of Real-Time PCR are as shows:the mRNA expression are up-regulated after transfection with these two plasmids, peaked on24h, and descended on36h,48h,72h in a time-dependent manner, though higher than normal Oh group.(4) The results of Western blot are as shows:the E-cadherin protein expression of these two plasmids transfection cells are both ascended on24h, peaked on36h, lower on48h and72h than36h, though higher than oh group. Nevertheless the E-cadherin protein expression in empty plasmid transfection group are a little lower on72h, while the α-SMA protein expression are higher than48h.Conclusion:(1) The two plasmid all have the potential to upregulation the expression of E-cadherin in Peritoneal mesothelium cell.(2)pIRES2-EGFP-ECAD is better than GV143-EGFP-ECAD to evaluate the Transfe-ction efficiency by the expression of EGFP, and Provide the experimental conditions for further experiments. This article contains figures12,ltables,...