Construction Of Vector Of Transgenic Mice Model And Expression Of Vector In Human Liver Cancer HepG2 Cells

Objective To construct vector oftransgenic mice model,test its feasibility of vector expression cassette and detect the expression and function(the effect on proliferation, apoptosis and cell cycle of the cell)of carried fat-1 gene in the eukaryote cell(human liver cancer HepG2 cells).In order to identify the suitability of vector.Methods Polyunsaturated fatty acids dehydrogenase fat-1 cDNA was inserted into eukaryotic expression vector pcDNA3.1(+)myc-His A to form a recombinant vector and transfected into HepG2 cell by liposomal reagent.The expression of fat-1 gene was detected by RT-PCR.MTT assay was applied to examine the effect of fat-1 gene on the proliferation of HepG2 cells.Gas chromatography was employed to examine the effect of fat-1 gene on the change in n-6/n-3 PUFAs ratio of HepG2 cells.And flow cytometry was used to examine the effect of fat-1 gene on cell cycle and apoptosis of HepG2 cells.Results The proposed recombinant plasmid was got through DNA recombinant technique;fat-1 gene effectively expressed in human liver cancer HepG2 cells;fat-1 mRNA band appeared 48h after transfection of pcDNA3.1(+)myc-His A-fat-1.Compared with the control cell(pcDNA3.1(+)myc-His A-control),proliferation of HepG2 cells was markedly inhibited(70%,p＜0.01) and apoptosis was promoted and cell cycle was mainly blocked in G₀/G₁ phase;Moreover,fat-1 gene significantly decreased ratio of n-6 /n-3 PUFAs of HepG2 cells membrane.Consequently,fat-1 gene could heterologously express in human liver cancer cells HepG2 and inhibit proliferation of cells by decreasing the ratio of n-6/n-3 PUFAs of cell membrane.It shows that the function of fat-lgene performs very well.Conclusions The constructed eukaryotic expression vector pcDNA3.1(+)myc-His A-fat-1 can be used as eukaryotic expression system.Carried fat-1 gene functions normally,reduces the n-6/n-3 PUFAs ratio of HepG2 cells,promotes the majority of HepG2 cells apoptosis in G₀/G₁ phase and inhibits their proliferation through a certain mechanism.Recombinant vector pcDNA3.1(+)myc-His A-fat-1 can provide a suitable vector for transgenic mice model.