Genetic modification of liver grafts with an adenoviral vector encoding the Bcl-2 gene improves organ preservation.
Genetic modification of liver grafts with an adenoviral vector encoding the Bcl-2 gene improves organ preservation.
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DOI：
10.1097/00007890-199903270-00001
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发表时间：
1999-03
期刊：
影响因子：
6.2
通讯作者：
G. Bilbao;J. Contreras;J. Gómez-Navarro;D. Eckhoff;Galina V Mikheeva;V. Krasnykh;Tracy Hynes;F. Thomas;Judith M. Thomas;D. Curiel
中科院分区：
文献类型：
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作者：
G. Bilbao;J. Contreras;J. Gómez-Navarro;D. Eckhoff;Galina V Mikheeva;V. Krasnykh;Tracy Hynes;F. Thomas;Judith M. Thomas;D. Curiel
背景：移植后肝功能的好坏取决于供体器官的质量以及保存、冲洗和再灌注损伤的影响。在这方面，细胞死亡（凋亡）在器官保存和排斥中起着重要作用。因此，我们研究了用编码Bcl-2基因的重组腺病毒载体对肝移植物进行遗传修饰以减少保存期间细胞凋亡的可能性。方法C57 B1/6小鼠的肝移植物采用标准技术获取和保存。本实验室成功构建了含人Bcl-2基因的复制缺陷型腺病毒载体（deltaE 1）（AdCMVhBcl-2）。使用编码不相关基因（大肠杆菌β-半乳糖苷酶）的腺病毒载体作为对照。每只小鼠在肝脏采集前48小时接受1 x 10（9）个空斑形成单位i. v.给药。对保存液中的肝酶活性进行分析。肝活检组织中的细胞凋亡通过DNA片段化和原位组织化学测定来确定。结果免疫组化和RT-PCR证实hBcl-2在移植物中表达。与对照组相比，表达hBcl-2的肝脏移植物的丙氨酸氨基转移酶（AST）和乳酸脱氢酶（LDH）的释放显着减少。复温后，在用AdCMVhBcl-2处理的动物的移植物中也观察到显著的细胞保护作用。组织学分析与保存液中转氨酶和LDH测定的肝细胞损伤相关。在表达hBcl-2的移植物中观察到凋亡细胞数量的显著减少。结论：我们已经证明了一种新的方法，以减少与人类Bcl-2基因保存损伤的肝移植。这种方法可以允许更长的保存时间，潜在地降低原发性无功能的发生率，降低冷损伤器官的免疫原性，并增加“边缘”肝移植物的安全使用。
BACKGROUND Liver function after transplantation is determined by the quality of the donor organ and the influences of preservation, flush, and reperfusion injury. In this regard, cell death (apoptosis) plays an important role in organ preservation and rejection. Therefore, we examined the possibility of genetic modification of the liver graft with a recombinant adenovirus vector encoding the Bcl-2 gene to reduce apoptosis during the preservation time. METHODS Liver grafts from C57B1/6 mice were procured and preserved using standard techniques. A replication defective adenovirus vector (deltaE1) containing the human Bcl-2 gene (AdCMVhBcl-2) was developed in our laboratory. An adenovirus vector encoding an irrelevant gene (Escherichia coli beta-galactosidase) was used as a control. Each mouse received 1 x 10(9) plaque forming units administered i.v. 48 hr before the liver procurement. Analyses of liver enzyme activities were determined in the preservation solution. Apoptosis in liver biopsies was determined by DNA fragmentation with an in situ histochemical assay. RESULTS Immunohistochemical analysis and RT-PCR confirmed the expression of hBcl-2 in the grafts. Grafts from livers expressing hBcl-2 showed significant reduction of the aspartame amino transferase (AST) and lactate dehydrogenase (LDH) release compared with grafts from the control groups. After rewarming, significant cytoprotection was also observed in grafts from animals treated with AdCMVhBcl-2. Histological analysis correlated with the hepatocellular injury determined with transaminases and LDH in the preservation solution. Significant reduction in the number of apoptotic cells was observed in grafts expressing hBcl-2. CONCLUSIONS We have demonstrated a novel approach to reducing the preservation injury to liver grafts with the human Bcl-2 gene. This approach may allow a longer preservation time, potentially reduce the incidence of primary nonfunction, decrease the immunogenicity of the cold injured organ, and increase the safer use of "marginal" liver grafts.