重组9型腺相关病毒介导细胞周期蛋白A2基因转染小鼠心肌的研究

Study of cyclin A2 transfected into mice myocardium mediated by recombinant adeno- associated virus 9

目的探讨重组9型腺相关病毒介导细胞周期蛋白A2基因经尾静脉注射后转染小鼠心肌的可行性及安全性。方法雄性C57BL／]~鼠随机分为病毒基因转染组和单纯生理盐水组。两组小鼠分别通过尾静脉注射病毒稀释液或等体积的生理盐水，分别于注射2周及4周后处死小鼠。Western印迹检测小鼠心脏、肝脏、肺脏和肾脏中细胞周期蛋白A2的表达，以评价腺相关病毒介导目的基因转染的靶向性。免疫组织化学检测细胞周期蛋白A2在心肌细胞中的表达及定位。检测增殖相关蛋白在各器官中的表达，明确转染后是否引起其他器官过度增殖，以评价其安全性。结果细胞周期蛋白A2转染2周后在小鼠心脏开始出现表达，第4周呈稳定表达，而对照组细胞周期蛋白A2的表达则相对较少（2周：0．13％±0．01％比25．97％±2．24％，4周：0．14％±0．02％比73．70％±3．10％），两组比较差异均有统计学意义（均为P〈0．05）。而两组小鼠肝脏、肺脏、肾脏中细胞周期蛋白A2的表达差异无统计学意义（均为P〉0．05）。细胞周期蛋白A2转染心肌后主要定位于心肌细胞胞质中，胞核中较少表达。转染心肌组织后，增殖细胞核抗原（PCNA）表达增加（2周：13．10％±0．54％比65．80％±3．44％，4周：12．90％±0．34％比71．20％±1．58％），两组比较差异均有统计学意义（均为P〈0．05）。而有丝分裂特异性蛋白磷酸化组蛋白H3（H3P）在两组中的表达差异无统计学意义（均为P〉0．05）。PCNA在肝脏、肾脏、肺脏中的表达差异无统计学意义（均为P〉0．05）。结论重组9型腺相关病毒介导细胞周期蛋白A2基因可靶向转染小鼠心肌，并能重新启动心肌细胞的细胞周期进程，且转染后不会引起其他器官的过度增殖，具有安全性及可行性。

Objective To explore the feasibility and safety of cychn A2 transfected into mice myocardium mediated by recombinant adeno-associated virus 9 through tail vein injection. Methods C57BL male mice were randomly divided into two groups, gene transfer group and control group. Mice were injected with 200 μl diluent of recombinant and saline respectively. Mice were killed two and four weeks after injection and hearts were harvested. Expression of cyclin A2 in heart, liver, lung and kidney were detected by Western blot, in order to evaluate the targeting of gene transfer. Location and expression of cyclin A2 in myocardium was checked by immunohistochemistry. Proliferation-related proteins were used to confirm whether excess multiplication existed after gene transfer, in order to evaluate the safety. Results Expression of cyclin A2 started at two weeks and obtained sustainable expression at four weeks. There were almost no expression in the control group （2 weeks ： 0.13%±0.01% vs 25.97%±2.24%, 4 weeks ： 0.14%±0.02% vs 73.70%±3.10% ） （all P〈0.05 ）. However, there were no significant differences of eyelin A2 expression in live, lung and kidney between two groups（all P 〉 0.05）. Location of cyclin A2 after gene transfer was mainly in cytoplasm and less in nuclear. Expression of proliferating cell nuclear antigen （PCNA） in myocardium was increased in gene transfer group （2 weeks： 13.10%±0.54% vs 65.80%±3.44%, 4 weeks： 12.90%±0.34% vs 71.20%±1.58%）, but did not change obviously in control group （all P〈0.05 ）. Phosphorylated histone H3 （H3P） in two groups had no significant difference （all P 〉 0.05 ）. Expression of PCNA in liver,lung and kidney showed no significant difference （all P 〉 0.05）. Conclusion Gene transfer of cyclin A2 via type 9 recombinant adeno-associated virus can restart the cardiomyocytes cell cycle, meanwhile, expression of cyclin A2 does not cause excess multiplication in other organs and this gene transfer method is safe and feasible.