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目的:构建磷酸受纳蛋白(PLB)反义RNA腺相关病毒载体(rAAV-asPLB),建立糖尿病(DM)大鼠模型。直接心肌注射rAAV-asPLB,观察其对DM大鼠心肌PLB基因转录和蛋白表达的影响,以及对心肌肌浆网Ca2+-ATPase活性的作用。方法:利用质粒辅助重组腺相关病毒系统试剂盒构建rAAV-asPLB。腹腔注射链脲佐菌素(STZ)诱导DM大鼠模型,将实验动物分为4组:正常组、DM组、盐水组和rAAV-asPLB组。盐水或rAAV-asPLB注射后6周,RT-PCR检测心肌PLBmRNA转录;Westernblotting检测PLB蛋白表达水平;检测心肌肌浆网Ca2+-ATPase活性。结果:(1)成功构建rAAV-asPLB,诱导出DM大鼠模型。(2)DM组和盐水组PLBmRNA水平均高于正常组;rAAV-asPLB组PLBmRNA水平较DM组和盐水组明显降低。(3)DM组和盐水组PLB蛋白水平均高于正常组;rAAV-asPLB组PLB蛋白水平较DM组和盐水组明显降低。(4)肌浆网Ca2+-ATPase活性在DM组和盐水组中较正常组明显降低,而rAAV-asPLB组较DM组和盐水组升高。结论:rAAV-asPLB抑制DM大鼠心肌PLB表达,增强Ca2+-ATPase活性。
OBJECTIVE: To construct the rAAV-asPLB antisense RNA of PLB (antisense RNA) and to establish a rat model of diabetes mellitus (DM). Direct injection of rAAV-asPLB into the myocardium to observe its effect on myocardial PLB gene transcription and protein expression in DM rats and its effect on myocardial sarcoplasmic reticulum Ca2 + -ATPase activity. Methods: The rAAV-asPLB was constructed by using the plasmid-assisted recombinant adeno-associated virus system kit. The diabetic rats were induced by intraperitoneal injection of streptozotocin (STZ). The experimental animals were divided into 4 groups: normal group, DM group, saline group and rAAV-asPLB group. Six weeks after saline or rAAV-asPLB injection, PLB mRNA transcription was detected by RT-PCR, PLB protein expression was detected by Western blotting, and Ca2 + -ATPase activity was detected in sarcoplasmic reticulum. Results: (1) rAAV-asPLB was successfully constructed and DM rat model was induced. (2) The levels of PLB mRNA in DM group and saline group were higher than those in normal group. PLB mRNA level in rAAV-asPLB group was significantly lower than that in DM group and saline group. (3) PLB protein levels in DM group and saline group were higher than those in normal group; PLB protein level in rAAV-asPLB group was significantly lower than DM group and saline group. (4) Ca2 + -ATPase activity in sarcoplasmic reticulum was significantly lower in DM group and saline group than in normal group, while rAAV-asPLB group was higher than DM group and saline group. Conclusion: rAAV-asPLB can inhibit the expression of PLB and enhance the activity of Ca2 + -ATPase.