The Effect Of ACE2 Gene Overexpression On Proliferation And Apoptosis Of Human Umbilical Vein Endothelial Cells Induced By Angiotensin â…¡

Objectives: To construct, package a lentiviral vector carrying ACE2 gene and observe the effect of ACE2 gene overexpression on proliferation reduced and apoptosis increased of human umbilical vein endothelial cells induced by Ang II after ACE2 gene lentiviral vector infected ,and to explore the possible mechanism.Methods: 1.ACE2 gene was amplified from plasmid pc-DNA3.1-hygro(+)-mACE2 by PCR technique and subcloned into the expression plasmid of pGC-FU lentiviraI vector.To generate the pGC-FU-ACE2 Lentiviral expression vector.The correct ACE2 gene was confirmed by endoenzyme digestion and sequencing . Recombinant Lentiviral-ACE2 were produced by 293T cells following the co-transfection of pGC-FU-ACE2 and packaging plasmids--pHelper1.0 and pHelper2.2. The ACE2 mRNA and protein levels of human umbilical vein endothelial cells were detected by RT-PCR and Western-blot techniques after 72 hours infection with multiplicity of infection of 10 (MOI = 10).3.The human umbilical vein endothelial cells were treated with Ang II (at final concentration 10-7mol/L) after infected by Lentiviral-ACE2, then were randomly divided into normal cells, AngⅡgroup, AngⅡ+Lentiviral-GFP group and AngⅡ+Lentiviral-ACE2 Group. The morphological changes of human umbilical vein endothelial cells in each group was observed by inverted phase contrast microscope;The proliferation function of each group human umbilical vein endothelial cells was detected by AlamarBlue reagents ;The apoptosis in each group human umbilical vein endothelial cells was researched by TUNEL Apoptosis Detection Kit,and to study Preliminary of the mechanism of ACE2 play the above roles.Results: 1. The recombinant lentiviruse Lentiviral-ACE2 was produced successfully,and the highly-purified virus liquid was obtained .2. Lentiviral-ACE2 can deliver target gene ACE2 to HUVEC,and can make the ACE2 express highly in human umbilical vein endothelial cells. 3. The cell growth state in AngⅡgroup and AngⅡ+ Lentiviral-GFP group were poor,slow growth, irregular shape and there were some degrees of cell suspension off wall; while the normal cell control group and group of AngⅡ+Lentiviral-ACE2 cell growth were in good condition, round,oval,full, and they were "paving stone"-like normal morphology growth and closely adherent,having little suspension cells.The AlamarBlue reduction rate of AngⅡgroup was significantly lower than normal cells (P<0.05),while the AlamarBlue reduction rate in AngⅡ+ AngⅡ+Lentiviral-ACE2 group was significantly higher than in AngⅡgroup(P<0.05);The apoptosis index between AngⅡgroup (0.1165±0.0181) and normal cells control group (0.0373±0.0113) had a clear statistical significance (P<0.05), besides, AngⅡgroup apoptosis index compared to AngⅡ+Lentiviral-ACE2 Group apoptosis index (0.0540±0.0061) had also a clear statistical significance (P<0.05).The AlamarBlue reduction rate and apoptosis index between AngⅡgroup and AngⅡ+Lentiviral-GFP was not statistically significant , nor between normal cells control group and AngⅡ+Lentiviral-ACE2.Conclusion: The recombinant lentiviruse Lentiviral-ACE2 was produced successfully and it can deliver target gene ACE2 to human umbilical vein endothelial cells; Ang II has a function that it can reduce the proliferation and increase the apoptosis of human umbilical vein endothelial cells,while the overexpression of ACE2 gene can restrain those actions.