关键词: 基因, 动脉粥样硬化, 血管紧张素转化酶2, 克隆

Abstract:

Objective  To construct replication-deficient recombinant adenovirus Ad-ACE2 and to investigate the effects of angiotensin-converting enzyme 2 (ACE2) on the severity of atherosclerosis in apolipoprotein-E knockout (ApoE-/-) mice. Methods  The full-length ACE2 encoding sequence was amplified from the RNA of mice kidney tissue by RT-PCR technique, cloned into plasmid pMD18-T vector, and then subcloned into plasmid pDC316 to form pDC316-ACE2. Homologous recombination was conducted between the shuttle plasmid and adenovirus skeleton plasmid to form recombinant adenovirus plasmid, then recombinant adenovirus plasmid was packed into replication-deficient recombinant adenovirus (Ad-ACE2) in the 293 cell. High-fat feeding was applied to establish 16 mice models of atherosclerosis, which were then divided into two groups randomly, receiving Ad-ACE2 and Ad-EGFP tail vein injection respectively. The lipid contents, protein expressions of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin were evaluated by Oil Red O staining, immunohistochemical method and Western blotting. Results  The recombinant plasmid Ad-ACE2 was confirmed by polymerase chain reaction, enzyme digesting and DNA sequencing. The lipid contents, protein expressions of VCAM-1 and E-selectin were significantly lower in ACE2 gene treatment group than in ACE2 gene control group. Conclusion  Ad-ACE2 is constructed successfully. Overexpression of ACE2 gene can reduce the lipid contents and protein expressions of VCAM-1 and E-selectin in the atherosclerotic plaque, and alleviate the severity of atherosclerotic plaque in ApoE-/- mice.

Key words: Clone, Atherosclerosis, Angiotensin-converting enzyme 2, Gene