Establishment Of VEGF-Transfected HUVECs Lines And Their Effect On The Proliferation Of VSMCs

The cardiovascular and cerebrovascular diseases caused by atherosclerosis(AS) arethe leading causes of morbidity and mortality around the world. The rapid developmentand widespread use of various vascular stents offers hope for AS patients. In-stentrestenosis after stent implantation, however, is an unresolved problem that limitstherapeutic efficacy. Although the clinical use of drug-eluting stents in recent years hasresulted in a significant reduction in the rate of restenosis, restenosis remains a problem.Because drug-eluting stents induce non-specific antiproliferative effects not only onvascular smooth muscle cell but also on other cell types such as endothelial cells,delaying vascular re-endothelialization process after surgery, further resulting inrestenosis.The interaction between endothelial cells and smooth muscle cells is the basis forthe development of cardiovascular diseases, the endothelial cells layer of the vessel wallintima is very susceptible to damage in the process of stent implantation.After injury ofthe vessel wall, vascular smooth muscle cells from the static contractile phenotypedifferentiate into synthetic phenotype which benefit of migration, proliferation,ultimately leading to vascular restenosis. Therefore, accelerating there-endothelialization of intravascular stents and rapidly restoring the integrity andfunction of the endothelium could prevent restenosis.In this research, cells used in tissue engineering were obtained which would beseeding cells implanted on stent. The seeding cells on the stent surface be able toaccelerate endothelial cells proliferation, migration and speed of endothelialization,which could inhibit proliferation of smooth muscle cells, and further prevent stentrestenosis. In addition, the Transwell co-culture model is used to culture endothelialcells and rat vascular smooth muscle cells, and study effect of stabe expressing VEGF121endothelial cells on the proliferation of vascular smooth muscle cells under theregulation of co-culture. The results of full research as follows:According to the design principles of shRNA, two pSilencer3.1-ofVEGF121-shRNA eukaryotic interference expression vector and a negative interferencevector were constructed, and stable transfected cells choose by hygromycin. Moreover,endothelial cell with a stable expression of pIRES2-EGFP-VEGF121plasmid wasrecoveried, expression of VEGF protein and VEGF gene in the seeding cells were detected by Western blotting and RT-PCR. While screening out the pSilencer3.1-ofVEGF121-shRNA2expression plasmid which has the highest inhibition efficiency. TheHUVECs lines with the stable expression different protein and gene level of VEGF121has been established successfully.The proliferation rate stable transfection of VEGF gene endothelial cells wassignificantly faster than the control group and VEGF interference group by MTT assay,and the capacity of blood vessel formation and migration of cells was significantlyhigher than the control group and VEGF interference group. Compared with the controlgroup and the interference group, the NO secretion of transfected VEGF gene cells wassignificantly increased, and the skeleton of endothelial cells was stained withFITC-Phalloidin. The results founded that the F-actin filaments of the cytoplasm andactin stress fibers of VEGF interference group decreased significantly, the F-actin at theouter edges of cells also decreased significantly, The results indicated that theoverexpress VEGF vascular endothelial cells can promote the formation of thecytoskeletonThe primary rat smooth muscle cells were successfully cultured and identified bythe organization adherence method, while endothelial cells and vascular smooth musclecells were using co-cultured by Transwell co-culture system. The results founded thatstable expression of VEGF in endothelial cells could inhibit synthesis of DNA andprotein of vascular smooth muscle cells under co-culture conditions.In a world, the in vitro experiments confirmed that transfected VEGF geneendothelial cells not only help to promote proliferation, migration of endothelial cells,and accelerate endothelial repair, but also were able to inhibit proliferation of smoothmuscle cells. The grafts with genetically modified cells which could repair damagedendothelial cells might become a new powerful method for treatment of atherosclerosisand restenosis in the future.