Effects Of Canstatin Gene Transfection On Vasculogenic Mimicry In Hepatocellular Carcinoma Cell Line Hepg2 In Vitro And In The Formation Of A Very Possible Mechanism

Object:Hepatoma cell line HepG2 in vitro culture, to observe the formation of vascular mimicry, and to observe and explore the gene expression product of recombinant human Canstatin hepatoma HepG2 cells on vasculogenic mimicry in the formation of a very possible mechanism.Methods:pcDNA3.1-Canstatin-3Flag secreted recombinant plasmid transfection with Cacl2 into E.coli DH5a, selected by AMP, picked plasmid restriction endonuclease digestion, sequencing, lipid vector-mediated method with pcDNA3.1- Canstatin-3Flag transfected into human hepatoma HepG2 cells, and use the empty vector pcDNA3.1 transfected hepatoma HepG2 cells as control, G418 medium screened positive clones, Western-blot identification of transfected cells Canstatin protein. The establishment of artificial basement membrane matrix gel HepG2 cells in vitro three-dimensional cell culture model, HepG2 cells were cultured in six-well plate as a control on the formation of vasculogenic mimicry in HepG2 ability to detect. To HepG2 group, empty vector transfected HepG2 group, recombinant vector was transfected into HepG2 cells were routinely cultured in 96 well plate 48h, MTT assay inhibition rate of value added in each group. To HepG2 group, empty vector transfected HepG2 group, recombinant vector was transfected into HepG2 cells were in the artificial basement membrane matrix gel in vitro culture, and compared three groups of cells forming ability of the pipeline, the formation of immunocytochemical detection of vascular mimicry The three groups of cells MMp-2, VE-cadherin protein expression in the case.Results:The successful person canstatin 684bp gene sequencing that in comparison with the same Genbank. PCR can detect the recombinant vector was transfected into human HepG2 cells genome contains genes Canstatin, Western-blot to detect Canstatin gene in recombinant plasmid transfected into HepG2 cells with specific expression. Human hepatoma HepG2 cells cultured under in vitro culture 72h, elongated spindle cells can be observed, many pseudopodia grow around each other and form a network connection, a ring or tube in human hepatocellular carcinoma HepG2 cells were cultured in six-well plate under adherent growth, can not form a ring or tube. To HepG2 group, empty vector transfected HepG2 group, recombinant vector was transfected into HepG2 cells were routinely cultured in 96 well plate 48h, MTT assay inhibition rate of value added in each group were:0%,3.57%,14.80%. To HepG2 group, empty vector transfected HepG2 group, recombinant vector transfected HepG2 group while in vitro culture,72h after the three cells can form tubular structures, select the VM under a microscope relatively concentrated area,200 times the microscope counting, re- vector transfection group structure The number of VM (2.67±2.08) were lower than the parental HepG2 cell group (9.67±3.51) months (P<0.01), empty vector transfected group The number of VM structures (11.00±3.61) a, and the parental HepG2 cell group (9.67±3.51) showed no significant meaning (P> 0.01). Recombinant vector transfected cells expression of VE-cadherin gray value (5.63±0.42) lower than the empty vector group (14.58±0.72) and HepG2 cell group (16.73±1.32) (P<0.01). HepG2 group, empty vector transfected HepG2 group, recombinant vector was transfected into HepG2 group MMp-2 protein expression in gray values were 27.53±1.03,25.72±0.83,27.02±2.13 (P> 0.01).Conclusion:1. Human hepatoma HepG2 cells were cultured in vitro can not be formed under the structure of vasculogenic mimicry in vitro culture conditions can form vasculogenic mimicry2.Successfully pcDNA3.1-Canstatin-3Flag secreted recombinant plasmid was transfected into HepG2 cells, its typical and can be expressed protein Canstatin3.Canstatin protein inhibits human hepatoma HepG2 cells value4.Canstatin protein inhibits in vitro culture conditions in human hepatoma HepG2 cells the formation of vasculogenic mimicry5.Canstatin protein inhibit in vitro culture conditions, HepG2 cells the expression of VE-cadherin protein, but no effect on the expression of MMp2.