Effect Of PDIA3on The Proliferation And The Apoptosis Of The Colonic Mucosal Epithelial Cells In The Experiments

Objective:To study the effection of PDIA3on the proliferation and the apoptosis of the colonicmucosal epithelial cells in USBS.Methods:In the early days,we established USBS rats model,and analysis the colonic mucosa ofthe USBS rats、the control group and the sham-operated group by proteomic technologies.Wefound more than ten kinds of protein which are associated with colon compensatory,such asprotein disulfide-isomerase A3(PDIA3), pyruvate kinase M1/M2et al by proteomics.Based onthese,we build pcDNA3.1/hisB-hPDIA3eukaryotic expression vector firstly,and transfectPDIA3expression plasmid into SW480cells by the method of gene transfection in ourexperiments. Then we validate the expression of PDIA3in SW480cells by Western blotting anddetect the effect of PDIA3on the proliferation of SW480cells. Finally, we detect the effect ofPDIA3on the apoptosis of SW480cells.Results:Wevalidate that there are two differences between sequence between148bp to1694bpwhich is needed to be coloned and reference sequence by DNA sequencing. While thedifferences are both in the third codon, which don’t change the original encoding amino acids.Sowe believe that we construct the plasmid of pcDNA3.1/hisB-hPDIA3successfully. According todifferent dose of transfection and transfection time，we transfect pcDNA3.1/hisB-hPDIA3intothe SW480cells by lipidosome and we extract protein from colonic mucosal epithelial cells aftertransfection. Then we validate the expression of PDIA3in SW480cells by Western blotting. As aresult, there are corresponding protein expressions in four transfection groups, while the proteinexpression of the group of24h which is also the high dose transfection group is the most clearone of the all groups. According to the dose and time of the transfection above, we transfectpcDNA3.1/hisB-hPDIA3into the SW480cell,and then we detect the changes in the rate ofSW480cell’s proliferation and apoptosis by the method of MTT flow cytometry technologyrespectively. The result of MTT is that the OD values of the three transfection groups are all higher thanthe blank control group. And in the three experimental groups, the absorbance valueof the cellsincrease with the increasing of the transfection dose, and the proliferation of SW480cell is moreclear. The result of cell apoptosis is the cell apoptosis rates of the three experimental groups arehigher than the blank control group. And in the three experimental groups, the apoptosis rate andtransfection is directly proportional to the dose of transfection (P <0.01).Conclusion:1．We construct the plasmid of pc DNA3.1/hisB-hPDIA3successfully and the result of Westernblotting confirmed that the plasmid constructed is well expressed in SW480cells and the besttime and dose of transfection.2．That PDIA3which is overexpressed can promote the proliferation of SW480cells and inhibitthe apoptosis effectively.