Various previous studies have found a negative cor-relation between the risk of cardiovascular events and serum high-density lipoprotein(HDL) cholesterol levels. The reverse cholesterol transport, a pathway of choles-...Various previous studies have found a negative cor-relation between the risk of cardiovascular events and serum high-density lipoprotein(HDL) cholesterol levels. The reverse cholesterol transport, a pathway of choles-terol from peripheral tissue to liver which has several potent antiatherogenic properties. For instance, the particles of HDL mediate to transport cholesterol from cells in arterial tissues, particularly from atherosclerotic plaques, to the liver. Both ATP-binding cassette trans-porters(ABC) A1 and ABCG1 are membrane cholesterol transporters and have been implicated in mediating cholesterol effluxes from cells in the presence of HDL and apolipoprotein A-I, a major protein constituent of HDL. Previous studies demonstrated that ABCA1 and ABCG1 or the interaction between ABCA1 and ABCG1 exerted antiatherosclerotic effects. As a therapeutic approach for increasing HDL cholesterol levels, much focus has been placed on increasing HDL cholesterol levels as well as enhancing HDL biochemical functions. HDL therapies that use injections of reconstituted HDL, apoA-I mimetics, or full-length apoA-I have shown dramatic effectiveness. In particular, a novel apoA-I mi-metic peptide, Fukuoka University ApoA-I Mimetic Pep-tide, effectively removes cholesterol via specific ABCA1 and other transporters, such as ABCG1, and has an an-tiatherosclerotic effect by enhancing the biological func-tions of HDL without changing circulating HDL choles-terol levels. Thus, HDL-targeting therapy has significant atheroprotective potential, as it uses lipid transporter-targeting agents, and may prove to be a therapeutic tool for atherosclerotic cardiovascular diseases.展开更多
Objective: To construct recombinant adeno-associated virus type 2(rAAV2) vectors encoding human apoA-I/apoA-lMilano protein, and explore an effective and safe method to prevent and treat the atherosclerotic disease...Objective: To construct recombinant adeno-associated virus type 2(rAAV2) vectors encoding human apoA-I/apoA-lMilano protein, and explore an effective and safe method to prevent and treat the atherosclerotic diseases. Methods: Human apoA-I cDNA with a His-tag in the upward stream of cDNA sequence were obtained with RT-PCR and PCR, and human apoA-IMilano cDNA was prepared by QuikChange Site-Directed Mutagenesis Kit. After extracted rAAV vectors with a most economic and convenient method, the particle numbers of rAAV vectors were measured by Dot-blot, and the purity was assayed by SOS-Page. The expression efficiency of the apoA-I or apoA-IMilano in C2C12 infected by rAAV vectors were detected by ELISA method. Results: ApoA-I cDNA was gained by RT-PCR and a His-tag was added in the upward stream of apoA-I cDNA successfully. ApoA-I cDNA was mutanted to apoA-IMilano cDNA successfully by QuikChange Site-Directed Mutagenesis Kit. The both titres of the rAAV vectors of apoA-I and apoA-IMilano were about 2×10^14/L, and the result of SOS-Page showed that the purity of the rAAV vectors was satisfied. The expression level of apoA-I was (0.39±0.04) μg/ml and the apoA-IMilano was (0.31±0.03) μ/ml in the DMEM culture medium at the first 24h after transfection. Conclusion: The success of the rAAV vectors construction, purification and the expression of apoA-I and apoA-IMilano in C2C12 cells mediated by these vectors, makes possible to inject rAAVA and rAAVAM vectors into mice muscle, and rises a new hope on finding a new way to prevent and treat atherosclerotic diseases and cardiovascular disease.展开更多
文摘Various previous studies have found a negative cor-relation between the risk of cardiovascular events and serum high-density lipoprotein(HDL) cholesterol levels. The reverse cholesterol transport, a pathway of choles-terol from peripheral tissue to liver which has several potent antiatherogenic properties. For instance, the particles of HDL mediate to transport cholesterol from cells in arterial tissues, particularly from atherosclerotic plaques, to the liver. Both ATP-binding cassette trans-porters(ABC) A1 and ABCG1 are membrane cholesterol transporters and have been implicated in mediating cholesterol effluxes from cells in the presence of HDL and apolipoprotein A-I, a major protein constituent of HDL. Previous studies demonstrated that ABCA1 and ABCG1 or the interaction between ABCA1 and ABCG1 exerted antiatherosclerotic effects. As a therapeutic approach for increasing HDL cholesterol levels, much focus has been placed on increasing HDL cholesterol levels as well as enhancing HDL biochemical functions. HDL therapies that use injections of reconstituted HDL, apoA-I mimetics, or full-length apoA-I have shown dramatic effectiveness. In particular, a novel apoA-I mi-metic peptide, Fukuoka University ApoA-I Mimetic Pep-tide, effectively removes cholesterol via specific ABCA1 and other transporters, such as ABCG1, and has an an-tiatherosclerotic effect by enhancing the biological func-tions of HDL without changing circulating HDL choles-terol levels. Thus, HDL-targeting therapy has significant atheroprotective potential, as it uses lipid transporter-targeting agents, and may prove to be a therapeutic tool for atherosclerotic cardiovascular diseases.
文摘Objective: To construct recombinant adeno-associated virus type 2(rAAV2) vectors encoding human apoA-I/apoA-lMilano protein, and explore an effective and safe method to prevent and treat the atherosclerotic diseases. Methods: Human apoA-I cDNA with a His-tag in the upward stream of cDNA sequence were obtained with RT-PCR and PCR, and human apoA-IMilano cDNA was prepared by QuikChange Site-Directed Mutagenesis Kit. After extracted rAAV vectors with a most economic and convenient method, the particle numbers of rAAV vectors were measured by Dot-blot, and the purity was assayed by SOS-Page. The expression efficiency of the apoA-I or apoA-IMilano in C2C12 infected by rAAV vectors were detected by ELISA method. Results: ApoA-I cDNA was gained by RT-PCR and a His-tag was added in the upward stream of apoA-I cDNA successfully. ApoA-I cDNA was mutanted to apoA-IMilano cDNA successfully by QuikChange Site-Directed Mutagenesis Kit. The both titres of the rAAV vectors of apoA-I and apoA-IMilano were about 2×10^14/L, and the result of SOS-Page showed that the purity of the rAAV vectors was satisfied. The expression level of apoA-I was (0.39±0.04) μg/ml and the apoA-IMilano was (0.31±0.03) μ/ml in the DMEM culture medium at the first 24h after transfection. Conclusion: The success of the rAAV vectors construction, purification and the expression of apoA-I and apoA-IMilano in C2C12 cells mediated by these vectors, makes possible to inject rAAVA and rAAVAM vectors into mice muscle, and rises a new hope on finding a new way to prevent and treat atherosclerotic diseases and cardiovascular disease.