| Objective: With As2O3,Canstatin and both joint scheme respectively intervention three-dimens ionalcultured HepG2cells, observe the effect of various factors on the intervention of vasculargenic mimicry(VM) forming ability (VM number, length and area), and to explore its impact mechanism, provide a newstrategy for cancer of the liver vascular resistance and experiment basis.Methods:1,CCK-8assay detect the proliferation inhibition rate of As2O3(1,5,10,15,20μmol/L) onHepG2cells;2, Identification of recombinant adenovirus;3,FCM detect the infection rates of differentmultiplicities of infection (MOI=10,20,40,60,80) adenoviral transfection of HepG2cells (24h,48h,72h),western-blot detect the expressing of Canstatin and Caspase-3in transfected cells;4,In vitro Matrigelthree-dimensional culture of HepG2cells, PAS staining to identify VM;5, The experiment of impact factoron VM formed by HepG2cells were divided into control group, As2O3group (1/2IC50, IC50,2IC50),Canstatin group, As2O3(1/2IC50, IC50) and Canstatin combination group, cell growth and VM formationwas observed under the microscope, photographed at three-dimensional cultured0.5,2,12,24,48and72h,the number,length and area of VM(ring quantity structure) were measured;6, RT-PCR to identify theexpression of VE-cadherin, MMP-2,Caspas-3and PCNA;7,Western-blot to detect the change of differentgroup in the expression of VE-cadherin, MMP-2,Caspase-3and PCNA protein.Results:1, Through the SPSS17.0statistical software to draw the72h half inhibitory concentration IC50(including10μmol/L) as the Dosage of follow-up experiments;2, PCR electrophoresis figure size of476bpbanding, sequencing of sequence recognition is Canstatin genes;3,2times,48h, MOI=80for the best wayto infection. Canstatin strongly expressed in transfection cells and no significance difference in theexpression of Caspase-3by Western blotting testing;4,2h after the start of the cell types of plasticdeformation, elongation and interconnected, with time, at12h mutually adjacent cells aggregate intodistinct cord-like structure, The structure gradually smooth edge and began to pile grow up at72h, PASstaining of ring cord-like structures formed by HepG2cells were dyed purple;5,Different options to dealwith3D cultured cells, the application of IPP software to calculate length, number, surface area of50xvision picture by inverted microscope. The results showed that, As2O3inhibit the formation of VM,inhibitory role of IC50group is stronger than1/2IC50group,P <0.05;The combined group can inhibit VM,P <0.05, but the difference between combination group and As2O3group were not statisticallysignificant,P>0.05; no significant difference between Canstatin group and the control group P>0.05;6,Theexpression of VE-cadherin,MMP-2,Caspas-3and PCNA can be tested;7, As2O3group treated cells after12h,1/2IC50group relative expression of VE-cadherin was1.79±0.13, IC50group was0.62±0.06,2.56±0.13in the control group and the difference was statistically significant P <0.05,1/2IC50group withIC50group relative expression of MMP-2of1.96±0.09,1.10±0.03and2.79±0.13in the control groupcompared to the difference was statistically significant P <0.05, whereas no difference in apoptosis-relatedprotein Caspase-3and the relative expression of proliferation-associated protein PCNA P>0.05, Canstatinrelative expression of proteins in each group with the control group than the difference was not statisticallysignificant,P>0.05, the combined group could inhibit the expression of VE-cadherin, but no difference incomparison with As2O3monotherapy group, has no effect on the expression of Caspase-3and PCNA.Conclusions:1, HepG2cells in vitro three-dimensional culture conditions have the ability to form VM;2,As2O3group of VM inhibition may be related to As2O3inhibited the expression of VE-cadherin andMMP-2;3, The joint group also inhibit the VM group, but no significant difference in comparison with theeffect of As2O3group;4, Canstatin had no effect on the expression of Caspase-3and VM. |
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