腺病毒介导的外源基因转移至供心的实验研究

目的探讨大鼠心脏移植过程中,重组腺病毒介导的外源基因转移至供心的可行性及安全性。方法取健康雄性Wistar大鼠140只,10周龄左右,体重200-250g。平均分为供、受体各70只,受体大鼠又随机分为重组腺病毒载体（recombinant adenoviral vector encoding the β-galactosidase gene.Ad-LacZ）基因转移组35只,对照组35只。手术制备建立同系大鼠腹腔异位心脏移植模型,在取出供心后,经供心冠脉循环系统于低温下（4℃）将Ad-LacZ 800μl灌注入供心,30min后进行异位心脏移植;对照组则灌注等量生理盐水。分别在移植术后3、5、7、14及28d取供心,经X-gal染色检测Ad-LacZ基因的表达情况和规律。术后第28天同时取受体移植心、肝、肾、肺和自体心脏组织进行组织学观察,并以腺病毒E1A区引物行组织RT-PCR检测,验证腺病毒是否具有复制可能性。结果在Ad-LacZ基因转移组移植心脏内检测到Ad-LacZ基因的表达,对照组未检测到Ad-LacZ基因的表达;Ad-LacZ基因转移组基因表达的高峰时间为术后3、5及7d（平均LacZ阳性染色细胞数/切片分别为66.4±23.1,91.3±32.4,68.7±22.7）组间比较,无统计学意义（P〉0.05）;14d（32.1±13.9）后逐渐下降,与3、5、7d时比较有统计学意义（P〈0.05）;28d（3.9±3.4）仅有极少表达,与3、5、7及14d时比较有统计学意义（P〈0.05）。术后28d各组受体重要脏器及自体心脏组织学检查无明显变化,RT-PCR扩增未见腺病毒E1A区mRNA产物。结论在心脏移植中,经供心冠状动脉系统以重组腺病毒为载体进行基因转移可以将目的基因有效且安全地转入供心内。

EXPERIMENTAL STUDIES OF ADENOVIRAL-MEDIATED EXOGENOUS GENE TRANSFER TO DONOR HEART

OBJECTIVE: To study efficiency and security of the recombinant adenoviral-mediated gene transfer to the donor heart during the heart transplantation. METHODS: A total of 140 healthy male Wistar rats, aged 10 weeks, weighing 200-250 g, were equally divided into the donor group and the recipient group, and then 70 rats in the recipient group were randomly and equally divided into 2 subgroups: the gene transfer group and the control group. The rat model of heterotopic heart transplantation(Abdomen)was developed, the donor hearts were removed and their coronary arteries were perfused with 800 microl of the recombinant adenoviral vectors encoding the beta-galactosidase gene (Ad-LacZ). The grafts were stored in the 4 C cold saline solution for 30 minutes, and then the syngeneic transplant was performed. In the control group, saline of tales doses was perfused. The donor hearts were harvested at 3, 5, 7, 14, and 28 days (n = 7) after transplantation, and the beta-galactosidase activity was assessed by the X-gal staining. At 28 days the major organs of the recipients were tested by the histopathological analysis and the polymerase chain reaction of the adenoviral E1A sequences. RESULTS: The successful gene transfer of the beta-galactosidase gene was demonstrated in the adenovirus-perfused hearts, with no staining in the control group. The gene expression reached a peak level at 3, 5 and 7 days, and the averaged numbers of the total beta-galactosidase positive staining cells per slice were 66.4 +/- 23.1, 91.3 +/- 32.4 and 68.7 +/- 22.7, respectively, with no significant difference between the groups (P > 0.05). At 14 days the gene expression gradually declined (32.1+/-13.9), and the significant difference was found when compared with that at 3, 5 and 7 days (P< 0.05). At 28 days the cells positive for beta-galactosidase were sparse (3.9 +/- 3.4), and the gene transfer was significantly less efficient compared with that at 3, 5, 7 and 14 days (P<0.05). The major organs of the recipients were not affected seriously at 28 days. No virus spread to other organs in this experimental protocol. CONCLUSION: The ex vivo adenoviral-mediated gene transfer intracoronarily to the donor heart during the heart transplantation is feasible and safe.