The Study Of Gene Construction Of Adeno-Associated Viral Vector-Mediated Vascular Endothelial Growth Factor 121 And Its In Vitro Expression On Human Umbilical Vein Endothelial Cells

Objective The purpose of this study was to demonstrate the changes ofcultured Human Umbilical Vein Endothelial Cells(HUVEC) which were infectedby the recombinant Adeno-Associated Viral vector(rAAV) expressing humanVascular Endothelial Growth Factor gene-121(VEGF₁₂₁),and evaluate theangiogenesis effect of VEGF₁₂₁ gene on vascular endothelial cells in vitro.Methods 1.The human VEGF gene was obtained by reversetranscription-polymerase chain reaction(RT-PCR)and directly cloned into thepAAV-MCS,the recombinant plasmid was called pAAV-VEGF.2. To genenatethe pAAV-LacZ viral particles, using LacZ as a report gene, AAV-293 cells wasco-transfected with the three plasmids(pAAV-LacZ, pAAV-RC, and pHelper) bythe way of liposome-mediated transfection.Then genenated the pAAV-VEGFviral particles in the same condition. To confirm that the transfection wassuccessful and evaluate the transfection rate, AAV-293 cells containingpAAV-LacZ-derived virus was stained by In Situ β-Galactosidase Staining Kit(X-gal). To measure the titer of adeno-associated recombinant virus stockscontaining VEGF₁₂₁ indirectely, the recombinant AAV particles containingLacZ was harvested and diluted over a 10-fold series from 10^-2 to 10^-5 .ThenHT-1080 cells, a human fibrosarcoma cell line,was infected with these differentfolds of diluted viral stocks and stained by X-gal. 3. To establish the reseach cellmodles, HUVECs were infected with viral particles containing AAV-VEGF₁₂₁ andpAAV-MCS, respectively. Then performed the cell proliferation assay using CellCounting Kit-8 (CCK-8), determined the ultrastrcture change in cells under theelectron-microscope,and detected the expression levels of VEGF₁₂₁ mRNA bysemi-quantitave RT-PCR.Results 1. The recombinant AAV expressing human VEGF121 gene wassuccessfully constructed and confirmed by DNA sequencing. 2. The viral titer ofrecombinant AAV measured by staining of In Situ β-Galactosidase Staining Kitin HT1080 cells was 6.4×107 particles/ml,and the transfection rate was 20%. 3.Comparing to the control group, assay demonstrated that the cell proliferateability in the treatment group was higher.Ultrastructural observation in cellsdemonstrated that the quanity and activity of cytosols related to proteinsynthesis in the treatment group increased significantly, while many lysosomeswere similarly found in two groups. Semi-quantitave RT-PCR revealed thatVEGF121 mRNA expressed in the treatment group was higher significantly thanthat of the control one(P=0.000).Conclusion When co-transfecting AAV-293 cells with three plasmids,the titerof recombinant AAV-VEGF121 particles can be achieved up to 107 particle/mland more stably. The VEGF121 gene can be expressed effeciently in manyaspectes when using recombinant AAV viral to infect the cultured HUVECs. Thisstudy has verifieded the fact that AAV Vector-Mediated VEGF121 gene has theangiogenesis effect on vascular endothelial cells in vitro.