CAJ | 학술논문

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小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.
목적 탐토중조9형선상관병독개도세포주기단백A2기인경미정맥주사후전염소서심기적가행성급안전성.방법 웅성C57BL소서수궤분위병독기인전염조화단순생리염수조.량조소서분별통과미정맥주사병독희석액혹등체적적생리염수,분별우주사2주급4주후처사소서.Western인적검측소서심장、간장、폐장화신장중세포주기단백A2적표체,이평개선상관병독개도목적기인전염적파향성.면역조직화학검측세포주기단백A2재심기세포중적표체급정위.검측증식상관단백재각기관중적표체,명학전염후시부인기기타기관과도증식,이평개기안전성.결과 세포주기단백A2전염2주후재소서심장개시출현표체,제4주정은정표체,이대조조세포주기단백A2적표체칙상대교소(2주:0.13％±0.01％비25.97％±2.24％,4주:0.14％±0.02％비73.70％±3.10％),량조비교차이균유통계학의의(균위P ＜0.05).이량조소서간장、폐장、신장중세포주기단백A2적표체차이무통계학의의(균위P＞ 0.05).세포주기단백A2전염심기후주요정위우심기세포포질중,포핵중교소표체.전염심기조직후,증식세포핵항원(PCNA)표체증가(2주:13.10％±0.54％비65.80％±3.44％,4주:12.90％±0.34％비71.20％±1.58％),량조비교차이균유통계학의의(균위P＜0.05).이유사분렬특이성단백린산화조단백H3(H3P)재량조중적표체차이무통계학의의(균위P＞0.05).PCNA재간장、신장、폐장중적표체차이무통계학의의(균위P＞0.05).결론 중조9형선상관병독개도세포주기단백A2기인가파향전염소서심기,병능중신계동심기세포적세포주기진정,차전염후불회인기기타기관적과도증식,구유안전성급가행성.
Objective To explore the feasibility and safety of cyclin 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 cyclin 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.