Construction Of Recombinant Lentiviral Vector Of Tie2-RNAi And The Study Of It's Intervention Of Malignant Melanoma Cells In Vitro

Objective:To construct and identify lentivector carrying Tie2-small interfering RNA(SiRNA) for studying the role of it,s intervention of malignant melanoma cells. The study will supply the theory basis to the further animal research on the inhibition of tumor growth in vivo and provide the experimental evidence for tumor gene therapy in future.Methods:1,Using the restriction endonuclease XbaI to cut the recombinant plasmids of pSilencer1.0-U6-Tie2-siRNA for getting U6-Tie2-siRNA wich was identified by electrophoresis.2,Using the restriction endonuclease XbaI to cut the pNL-EGFP vectors and identifying it by electrophoresis.3,Connect the pNL-EGFP vectors and U6-Tie2-siRNA which were identified by using enzyme digestion electrophoresis of the restriction endonuclease XbaI, and the density of them is 1:10. And the Lentiviral transfer plasmids pEGFP-Tie2-â… and pEGFP-Tie2-â…¡were constructed, then identified by using enzyme digestion electrophoresis of the restriction endonuclease XbaI and sequencing analysis.4,Lentiviral vector systems of three plasmids were constituted using pEGFP-Tie2-â… ,pEGFP-Tie2-â…¡,pVSVG and pHelper,and the density of them is 1:1:1. The Lentiviral vector system was transfected into 293T cell to produce pEGFP-Tie2-â… ,pEGFP-Tie2-â…¡lentivirus.5,Collecting the virus supernatant and using the different density of the collected supernatant to determination the viral titer.6,To choice an appropriate infection plural number for further experiment by using different infection plural number of lentivirus to infect malignant melanoma cells.7,With appropriate infection plural number,malignant melanoma cells were infected by lentivirus and Realtime RT-PCR was conducted to examine inhibitory efficiency of the expression of Tie2 gene,and get the rate of the inhibition.Results:1,The U6-Tie2-siRNA was constructed successfully from the recombinant plasmids of pSilencer1.0-U6-Tie2-siRNA by using enzyme digestion electrophoresis of the restriction endonuclease XbaI.2,The lentiviral transfer plasmids of pEGFP-Tie2-â… and pEGFP-Tie2-â…¡were constructed successfully and identified by using enzyme digestion electrophoresis of XbaI and sequencing analysis. And the results are correct.3,lentivector carrying Tie2-small interfering RNA were produced by using the three plasmid lentiviral packaging system. The viral titers measured by 293T cells is 8.8x103/ul.4,When the infection plural number is 80, the infection efficiency is 94.5%,The results of Reatime RT-PCR demonstrated that the lentivector carrying Tie2-small interfering RNA could inhibit the expression of Tie2 genes in malignant melanoma cells, and the high rate of the inhibition was 68.55%(p<0.05), and there is no significant difference on the expression levels(P>0.05) between the two lentiviral vectors of Tie2-RNAi.Conclusions:Lentivector carrying Tie2-small interfering RNA(SiRNA) was constructed successfully by using the three plasmid lentiviral packaging system, and it was able to inhibit the expression of Tie2 gene of malignant melanoma cells, and it would be useful for the further experiment of the inhibition of the growth of nude mouse transplantable tumor, and it would provide experiment evidence for further experiment .