The Recombinant Plasmids Of PSilencer 1.0-U6-Ang2/Tie2-siRNA Were Constructed And The Function Of Them On Angiogenesis In Vitro

ObjectiveThe expression of Ang2 and Tie2 is silenced by RNA interference in order to inhibit the angiogenesis in vitro. The study will supply the theory basis to the further animal research on the inhibition of tumor angiogenesis in vivo and provide the experimental evidence for tumor gene therapy.Methods1,Recombinant pSilencer 1.0-U6 RNAi plasmids targeting the mRNA of Ang2,Tie2 were constructed and there are two recombinant plasmids for each gene.2,The protein and mRNA expression levels of Ang2 and Tie2 in HUVECs, transfeted by the recombinant plasmids,were analyzed by immunocellochemistry and RT-PCR in each groups respectively.3,the formation of vascular-like structure in HUVECs,transfected by the plasmids ,was studied in vitro by three-dimensional culture.Results1,The recombinant plasmids of pSilencer1.0-U6-Ang2/Tie2-siRNA were constructed successfully, and could not be digested by Hind III. The result of recombinant plasmids sequencing matchs that of the design.2,the protein and mRNA expression levels of Ang2,Tie2 in HUVECs ,transfected by the plasmids pSilencer 1.0-U6-Ang2/Tie2-siRNA, were suppressed by immunocytochemistry and RT-PCR(P<0.05), but there is no significant difference on the expression levels(P>0.05)between the two plasmids of each siRNA .3,There is a significant decrease on the quantity and length of vascular-like structure in HUVECs transfected with the plasmids by three-dimensional culture model in vitro,(P<0.05).It proves that angiogenesis has been inhibited significantly in vitro.Conclusions1,The plasmids of pSilencer1.0-U6-Ang2/Tie2-siRNA were constructed successfully.2,Ang2-siRNA,Tie2-siRNA can inhibit the protein and mRNA expression of Ang2,Tie2, and inhibit angiogenesis in the model of three-dimensional culture in vitro.