Wfdc1 Gene Cloning And Growth Inhibition Of Hepg2 Cells

PartⅠ:WFDC1 GENE CLONG AND THE CONSTRUCTION OF RECOMBINANT EUKARYOTIC EXPRESSION VECTOR pcDNA3.1(+)-WFDC1Objectives:TO Clone WFDC1 gene from normal human prostate tissue and construct Eukaryotic expression vector pcDNA3.1 (+)-WFDC1。Methods: Total mRNA was extracted from normal human prostate tissue, and specific primers were designed in order to clone human WFDC1 gene by RT-PCR method. Identified by sequencing, it was inserted into pcDNA3.1(+) Eukaryotic expression vector by HindⅢand EcoRⅠrestrict enzyme digestion. Results: the fragment length of WFDC1 gene was 684bp, it codes 221 amino acid(AA), molecular weight is 2.4kd, among them, there are 23 acid AA,30 basic AA,48 polar AA and 65 hydrophobic AA. Identified by sequencing and restrictive endonuclease digestion, the recombinant eukaryotic expression vector pcDNA3.1(+)-WFDC1 had been constructed correctly. Conclusions:The constructed recombinant Eukaryotic expression vector pcDNA3.1(+)-WFDC1 would lay down the basis for transfection PartⅡ:STABLE EXPRESSION OF WFDC1 GENE IN HEPG2 CELL AND STUDY OF WFDC1 GENE INHITITS HEPG2 CELL COLONY-FORMINGObjectives:To study the stable expression of transfected WFDC1 gene in HepG2 cell that contains in vitro and in vivo and the inhitition of The transfected of WFDC1 gene on the growth of hepatoma cell lines.Methods: 1,(conloy formation) HepG2 was transfected with pcDNA3.1(+)-WFDC1 recombinant vector named experimental group. HepG2 was transfected with pcDNA3.1 (+) were enrolled as control grorp. Two groups have 7 samples.Different plasmid was transfected into HepG2 with lipofection method.After transfection, observed the changes of cell morphology and identified the expression of WFDC1 gene by RT-PCR.Analysis the number of colony formation of two groups.2,(tumorigencity experiment) 18 nude mices randomly divided into 3 groups and inoculated with different cellssubcutaneously. Nude mices of experimental group were inoculated cell transfected pcDNA3.1(+)-WFDC1,control groups was inoculated HepG2 transfected with vector and HepG2,then the growth of transplanted tumor was observed.After 4 weeks, identified the expression of WFDC1 gene in there tumor tissue by RT-PCR. Results:1,In experimental grorp, maximum of colony is 46,minimum is 21,averagei is 34.4±8.8,in control group, maximum of colony is 60,minimum is 35,averagei is 45.7±8.2(p<0.05),there are significant difference between the two groups.The transfection of WFDC1 supressed the colony formation capability of transfected HepG2 cell.2,Three groups were grown tumor.The experimental grorp expresses WFDC1 gene by RT-PCR.The other groups were not express WFDC1 gene.Conclusions:Transfeected WFDC1 gene could satably expressed in HepG2 cell.The transfection of WFDC1 gene suppressed the growth and colony formation capability of HepG2 cell.The results is the basic of WFDC1 gene therapy for liver cancer.