Construction Of Recombinant Eukaryotic Expression Plasmid Containing Mice ACE2 Gene And Effect Of ACE2 Gene Transfection On Proliferation Of Vascular Smooth Muscle Cells

Objective: This study planned to construct eukaryotic expression vector containing mice ACE2 gene ,to observe the cell multiplication effect of Angâ…¡on rat vascular smooth muscle cell(VSMC) after ACE2 gene transfected ,and to approach the possible mechanism.Method:1. The full-length cDNA of mice ACE2 was amplified from mice kidney by RT-PCR.Cloned it into pcDNA3.1/Hygro(+) to construct eukaryotic expression vector pm-ACE2 containing mice ACE2 gene,and identified it by enzyme digestion and sequencing.2.Transfect pm-ACE2 into COS-7 cell by Lipofectamine 2000,then to authenticate the expression function in eukaryotic cell of pm-ACE2 by Western-Blot.3. Transfect pm-ACE2 into primary culture rat aorta pectoralis VSMC by Lipofectamine 2000 and dealed it with Angâ…¡(final concentration10-7mol/L).Normal cell group, Angâ…¡group and pcDNA3.1/Hygro(+) transfected+Angâ…¡group were as control groups.To observe the cell multiplication effect of Angâ…¡on rat vascular smooth muscle cell(VSMC) after ACE2 gene transfected by using CCK8 and cell cycle detection,and to approach the possible mechanism.Result: 1.Mice ACE2 gene was successfully cloned and linked to vehicle pcDNA3.1/Hygro(+).Sequencing result showed that there were three spot of samesense mutation.2. pm-ACE2 had expression function in eucaryotic cell.3. The OD value of Angâ…¡group were obviously higher than that of control group(P<0.05)and pm-ACE2+Angâ…¡group were obviously lower than the Angâ…¡group in OD value.4.To compare with the control group ,the G0/G1 stage percentage of VSMC in Angâ…¡group was obviously lower while the percentage of S stage was obviously higher(P<0.05).To compare with the Angâ…¡group , the G0/G1 stage percentage of VSMC in pm-ACE2+Angâ…¡group was obviously higher while the percentage of S stage was obviously lower(P<0.05).Conclusion: Eukaryotic expression vector of mouse ACE2 gene was successfully constructed. Angâ…¡has the function of promoting VSMC proliferation which can be restrained by ACE2 gene.