Bilbao G, Contreras J L, Gómez-Navarro J, Eckhoff D E, Mikheeva G, Krasnykh V, Hynes T, Thomas F T, Thomas J M, Curiel D T
Gene Therapy Center, Department of Surgery, University of Alabama at Birmingham, 35294, USA.
Transplantation. 1999 Mar 27;67(6):775-83. doi: 10.1097/00007890-199903270-00001.
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.
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.
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.
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.
移植后的肝功能取决于供体器官的质量以及保存、灌注和再灌注损伤的影响。在这方面，细胞死亡（凋亡）在器官保存和排斥反应中起重要作用。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基因改造以减少保存期间细胞凋亡的可能性。
采用标准技术获取并保存C57B1/6小鼠的肝移植物。我们实验室构建了一种含有人Bcl-2基因的复制缺陷型腺病毒载体（deltaE1）（AdCMVhBcl-2）。使用编码无关基因（大肠杆菌β-半乳糖苷酶）的腺病毒载体作为对照。每只小鼠在获取肝脏前48小时经静脉注射给予1×10⁹空斑形成单位。测定保存液中的肝酶活性。通过原位组织化学分析DNA片段化来确定肝活检组织中的细胞凋亡情况。
免疫组织化学分析和逆转录-聚合酶链反应证实了移植物中hBcl-2的表达。与对照组移植物相比，表达hBcl-2的肝脏移植物中天冬氨酸氨基转移酶（AST）和乳酸脱氢酶（LDH）的释放显著减少。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所确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显著减少。
我们已证明一种用人类Bcl-2基因减少肝移植物保存损伤的新方法。这种方法可能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“边缘性”肝移植物的安全使用。
