【摘要】 目的研究hBcl-2基因转染对大鼠肝脏抗缺血再灌注损伤的效应,并探讨其减轻移植肝缺血再灌注损伤的可行性。方法(1)同源重组法构建复制缺陷型重组腺病毒Adv-EGFP、Adv-Bcl-2,转染293细胞并包装成腺病毒颗粒后大量扩增和纯化。同法构建空载体病毒质粒;(2)雄性SD大鼠,随机分为Adv-Bcl-2转染组、Adv-EGFP转染组、缺血再灌注组、假手术对照组,以两袖套法建立同种异体肝移植模型。转染组经门静脉灌注转染重组腺病毒Adv-Bcl-2或Adv-EGFP;缺血再灌注组、假手术对照组不予任何处理。实时定量PCR和Western blotting检测各组肝组织bcl-2mRNA和蛋白表达变化;门静脉血流恢复后第0、60及180min三个时间点测定受体血清AST、LDH、MDA含量。组织切片检测肝细胞形态改变。结果 Adv-Bcl-2转染组bcl-2mRNA和蛋白表达水平较Adv-EGFP转染组和对照组明显增高(P<0.01);Adv-Bcl-2转染组中血清AST、LDH含量明显低于Adv-EGFP转染组和缺血再灌注组。Adv-Bcl-2组血清MDA水平明显低于缺血再灌注组和Adv-EGFP组(P<0.01)。与假手术组比较,Adv-Bcl-2处理组HE染色见少量肝细胞水肿和空泡变性,未见片状或点状坏死等。缺血再灌注组和Adv-EGFP组HE染色镜下观察可见小叶结构破坏、肝细胞弥漫不同程度水样变性、体积明显增大、细胞间边界模糊,还可见大量细胞出现脂肪变性及少量碎片状坏死。结论 Adv-Bcl-2转染能通过诱导bcl-2基因表达减轻移植肝缺血再灌注损伤。更多还原

【Abstract】 Objective To study the effect of hBcl-2 gene transfer on rat liver against ischemia-reperfusion injury,and explore the feasibility of this approach to reduce ischemia-reperfusion injury in liver transplantation. Methods We constructed the replication-deficient recombinant adenoviruses Adv-EGFP and Adv-Bcl-2 and transfected them into 293 cells and packaged into adenovirus particles for amplification and purification. The empty plasmid vector virus was constructed similarly. Male SD rats were randomized into Adv-Bcl-2-transfected group,Adv-EGFP-transfected group,ischemia-reperfusion group,and sham-operated group,and liver allograft transplantation model was established by sleeve method. In the transfected groups,the recombinant viruses were administered by perfusion through the portal vein,and the ischemia-reperfusion and sham-operated groups received no treatment. Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expressions of bcl-2 in the liver tissue of each group,and at 0,60 and 180 min after reperfusion,serum AST,LDH,and MDA levels were measured. Histological changes of the liver cells were evaluated by HE staining. Results Bcl-2 mRNA and protein expressions in Adv-Bcl-2-transfected group,as compared with those in Adv-EGFP-transfected group and control group,were significantly increased (P<0.01); the serum levels of AST,LDH and MDA in Adv-Bcl-2-transfected group were significantly lower than those of Adv-EGFP-transfected group and ischemia-reperfusion group (P<0.05 or 0.01). Compared with the sham-operated group,Adv-Bcl-2 treatment group showed lessened edema and vacuolar degeneration of the liver cells without patches or spots of necrosis. In ischemia-reperfusion and Adv-EGFP group,HE staining revealed hepatic lobular destruction and extensive liver cell swelling,enlargement,vacuolar degeneration,edema and occasional focal necrosis. Conclusion Adv-Bcl-2 transfection can induce the expression of bcl-2 gene to reduce ischemia-reperfusion injury of the liver graft in rats.更多还原