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Construction of phospholamban antisense RNA recombinant adenoassociated virus vector and its effects in rat cardiomyocytes

查看全文 作　　者：JiangLI,Shen-jiangHU JianSUN Zhao-huiZHU XiaZHENG Guo-zhongWANG Yu-meiYAO Nai-yunCHEN Xiao-[1]yanZHAO 高影响力作者 机构地区：[1]DepartmentofCardiology,FirstAffiliatedHospital,MedicalCollegeofZhejiangUniversity,Hangzhou310003,China高影响力机构 出　　处：《Acta Pharmacologica Sinica》索引2005年第26卷第1期,共5页高影响力期刊 摘　　要：Aim: To construct a recombinant adeno-associated virus (rAAV) vector containing gene encoding phospholamban antisense RNA (asPLB), and analyse its effect on expression of PLB, expression and activity of sarco-endoplasmic reticulum Ca^2+-ATPase (SERCA), and the change of intracellular free Ca^2+ concentration ([Ca^2+]i) in rat cardiomyocytes. Methods: The target gene encoding PLB antisense RNA was inserted inversely into the adeno-associated virus plasmid pAAV-MCS digested by corresponding restricted endonuclease enzyme. The recombinant plasmid and pAAV-RC and pHelper were co-transfected into 293 cell. At the same time, a viral production positive control (rAAV-LacZ) and negative control were performed. The recombinant viruses were used to transfect the cultured rat cardiomyocytes. Site β-Galactosidase staining were performed to observe the transfer efficiency. Reverse transcription-PCR and Western blot were used to determine the mRNA and protein expression of PLB and SERCA. The activity of SERCA and the [Ca^2+]i were measured. Results: The rAAV vectors were constructed successfully and were transfected into rat cardiomyocytes effectively.The PLB mRNA and protein expression were reduced in rat cardiomyocytes transfected by rAAV-asPLB compared with controls. The activity of SERCA was increased. In rest state, the level of [Ca^2+]i in the rAAV-asPLB transfected groupdecreased. The level of [Ca^2+]i increased when induced by isoproterenol.Conclusion: AAV-asPLB vector was constructed successfully, which disrupted the expression of PLB, enhanced the activity of SERCA, reduced the resting [Ca^2+]i, and improved the cardiac function. 关 键 词：磷脂酶 抗过敏RNA 重组腺病毒 心肌症 网状结构 肌奖钙 ATPASE
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