腺相关病毒载体介导的PLB基因反义RNA对大鼠心肌细胞肌浆网Ca2+-ATPase活性和［Ca2+］i的作用

目的：探讨腺相关病毒载体介导的磷酸受纳蛋白（PLB）反义RNA对肌浆网Ca2+-ATPase活性以及细胞内钙浓度（［Ca2+］i）的作用。 方法： 构建PLB反义RNA的重组腺相关病毒载体（rAAV-asPLB）和携带报告基因LacZ的重组腺相关病毒载体（rAAV-LacZ）。分别转染培养的大鼠心肌细胞，测定PLB mRNA和蛋白质表达，检测肌浆网Ca2+-ATPase活性和［Ca2+］i。 结果： RT-PCR和Western blotting显示，感染rAAV-asPLB的心肌细胞的PLB mRNA和蛋白质表达低于正常对照和感染rAAV-LacZ组；而Ca2+-ATPase活性大于正常对照和感染rAAV-LacZ组。激光共聚焦显微镜检测显示，静息状态时，rAAV-asPLB感染组［Ca2+］i降低；异丙肾上腺素刺激后，各组［Ca2+］i均升高，但是rAAV-asPLB感染组［Ca2+］i变化幅度大。 结论： 腺相关病毒介导的PLB反义RNA对大鼠心肌细胞PLB表达具有抑制作用，增强Ca2+-ATPase活性，降低静息状态的［Ca2+］i ，增加异丙肾上腺素刺激后［Ca2+］i 的变化幅度。

Effects of phospholamban antisense RNA on SR Ca2+ - ATPase and [ Ca2+ ]i in rat cardiomyocytes by adeno- associated virus vector

AIM: To investigate the effect of phospholamban antisense RNA (asPLB) on the activity of sarco-endoplasmic reticulum (SR) Ca 2 -ATPase, and the change of intracellular free Ca 2 concentration ([Ca 2 ]i) in rat cardiomyocytes by adeno-associated virus(AAV) vector. METHODS: rAAV-asPLB and rAAV-LacZ were constructed by AAV Helper-Free System. RT-PCR and Western blotting were used to determine the mRNA and protein expression of PLB. The activity of SR Ca 2 -ATPase and the [Ca 2 ]i were measured. RESULTS: Compared to controls, the PLB mRNA and protein expression reduced in rat cardiomyocytes transfected with rAAV-asPLB. The activity of Ca 2 -ATPase was increased. In rest state, the level of [Ca 2 ]i in rAAV-asPLB transfected group was decreased. The level of [Ca 2 ]i was increased when induced by isoproterenol. CONCLUSION: rAAV-asPLB vector disrupts the expression of PLB, enhances the activity of Ca 2 -ATPase, reduces the resting [Ca 2 ]i and enhances the isoproterenol-induced [Ca 2 ]i.