| [Objective]1,Three dimension(3D)model in vitro was constructed for angiogenesis to explore the effects of Ang1,2 and Tie2.The expression levels of Ang1,2 and Tie2 receptor of human umbilical vein endothelial cells(HUVECs),HUVECs in vitro and three-dimensional vascular-like structure in vitro was analyzed.2,The 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.[Methods]1,Collagenase was used to isolate the endothelial cell from human umbilical vein, (HUVECs),identified by immunocellochemistry.Three-dimensional vascular-like structure was constructed in vitro in the sandwich culture system.2,The mRNA and protein expression levels of Ang1,2 and Tie2 in 10 specimens of HUVECs in vivo,10 specimens of HUVECs in vitro,and 10 specimens of three-dimensional vascular-like structure in vitro were analyzed by RT-PCR,immunocellochemical and immunohistochemical methods respectively.3,Recombinant pSilencer 1.0-U6 RNAi plasmids targeting the mRNA of Ang2,Tie2 were constructed and there are two recombinant plasmids for each gene.4,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.5,the formation of vascular-like structure in HUVECs,transfected by the plasmids,was studied in vitro by three-dimensional culture.[Results]1,The mixed single cell suspension,most of which are HUVECs,was obtained from 1,The mixed single cell suspension,most of which are HUVECs,was obtained from human umbilical vein digested by Collagenase and purified by repeating cells plating. The identification of HUVECs is used by the technique of immunocellochemistry. Three-dimensional vascular-like structure was constructed in three-dimensional culture system.2,The protein and mRNA expressions of Ang2,Tie2 in three-dimensional vascular-iike structure in vitro are significantly higher than that in vivo and cultured in vitro.(P＜0.05).there is no significant difference on The protein and mRNA expression levels of Ang2,Tie2 between HUVECs in vivo and in vitro(P＞0.053,There is no significant difference on the protein of Ang1 and mRNA expression levels between HUVECs in vivo,in vitro and three-dimensional vascular-like structure in vitro respectively(P＞0.05)4,The recombinant plasmids ofpSilencerl.0-U6-Ang2/Tie2-siRNA were constructed successfully,-and could not be digested by HindⅢ.The result of recombinant plasmids sequencing matchs that of the design.5,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.6,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.[Conclusion]1,In vitro three-dimensional cell culture system is a convenient method which is suit for the growth of endothelial cell,the formation of tube-meshworks,the three-dimensional meshwork,and especially the study in the effects of the factors on the angiogenesis.2,The expression of Ang2 and Tie2 in vivo,in vitro and in vitro three-dimensional vascular-like structure are positive,but the expression levels of Ang2 and Tie2 in vitro three-dimensional vascular-like structure are significantly higher.It proves that Ang/Tie2 system takes an important part in angiogenesis.3,The plasmids of pSilencer1.0-U6-Ang2/Tie2-siRNA were constructed successfully.4,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.
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