【作者】 胡明政；

【导师】 钱海鑫；

【作者基本信息】 苏州大学 ， 外科学， 2007， 博士

【摘要】 背景:缺血再灌注损伤目前在肝移植中是不可避免地发生,并且可能导致移植肝出现原发性无功能,这也是临床中需要再移植的重要原因之一,因此重视对缺血再灌注损伤的研究有助于解决目前临床中供体紧缺的矛盾。而基因治疗是近年来迅速开展的临床治疗方法之一,利用基因转染技术来减缓移植肝的缺血再灌注损伤有着良好的临床应用前景。第一部分:大鼠原位肝移植模型的建立目的:用改良二袖套法建立稳定的大鼠原位肝脏移植模型。方法:在Kamada二袖套法的的基础上,对大鼠原位肝移植术在取肝、灌注、修肝和植肝等手术操作以及围手术期的处理进行改良,复制120例次大鼠原位肝移植模型。观察供、受手术时间、无肝期、手术成功率、术后并发症和一周存活率。结果:与训练初期相比较,训练的后期在手术时间、无肝期、手术成功率、术后并发症和移植后大鼠一周存活率均存在显著差异。结论:通过在Kamada二袖套法的基础上进行技术改进和一定数量的模型训练能够建立理想的大鼠原位肝移植模型。第二部分:携带hHO-1基因的重组腺病毒载体的构建目的:构建含有血红素氧合酶-1基因的重组腺病毒载体方法:(1)用限制性内切酶EcoRI+Bg1II从克隆载体pOTB7-hHO-1中切取基因hHO-1片段。(2)将线性化的pSGCMV质粒和经酶切的hHO-1基因进行连接转化,对其产物进行质粒鉴定,酶切鉴定和DNA测序鉴定。(3)采用Cre/Loxp系统将质粒pSGCMV-hHO-1和含5型腺病毒右臂的质粒pBGHLoxP△E1E3共转染至293细胞包装成重组腺病毒Ad-HO-1。(4)用VT001/VT002鉴定重组腺病毒Ad-HO-1的安全性,并用Ad-HO-1感染体外培养的细胞,观察其感染效率。结果:(1)利用连接酶将pSGCMV质粒和hHO-1连接转化可获得阳性质粒pSGCMV-hHO-1,并经鉴定目的基因hHO-1能准确完整地导入质粒pSGCMV中(。2)采用Cre/LoxP腺病毒重组系统能获得高滴度的重组腺病毒Ad-HO-1。(滴度为2.0×1010pfu/ml)。(3)重组腺病毒Ad-HO-1经VT001/VT002鉴定无野生型腺病毒,并能有效感染体外培养细胞使之表达hHO-1基因的产物。结论:采用Cre/LoxP腺病毒重组系统能有效地构建有良好安全性的携带hHO-1基因的腺病毒载体。第三部分Ad-HO-1对大鼠移植肝缺血再灌注损伤的保护作用目的:利用携带hHO-1基因的腺病毒载体研究其对大鼠移植肝缺血再灌损伤的保护作用。方法:SD大鼠32只配对成16对,随机分两组(n=8)行ROLT。对照组供体用空载体进行腹腔注射,实验组供体用Ad-HO-1进行腹腔注射。36小时后取肝行ROLT。缺血再灌注6小时后处死模型,采血送检肝功能;流式细胞仪测肝细胞凋亡率;Western-blot检测肝组织中的HO-1、Bcl-2、Bcl-xl和caspase-3,并进行灰度比较;TUNEL法检测肝组织的细胞凋亡;免疫组化检测HO-1、TNF-α、巨噬细胞和caspase-3;电镜观察肝细胞的超微病理变化。结果:(1)实验组的肝功能指标ALT、AST、LDH显著低于对照组;细胞凋亡率显著低于对照组,且凋亡细胞主要出现在汇管区附近。(2)Western-blot检测发现实验组肝组织中HO-1、Bcl-2和Bcl-xl显著高于对照组,而caspase -3则显著低于对照组。(3)免疫组化发现HO-1在实验组中大量表达且主要在血管区,而对照组中TNF-α在肝实质细胞大量表达,且血管壁附近可见大量巨噬细胞浸润,caspase -3阳性细胞出现于肝间质细胞。(4)电镜观察对照组可见典型细胞凋亡,而实验组肝细胞超微结构基本正常。结论:(1)构建携带hHO-1基因的腺病毒载体通过热转染能成功地转入供肝中并高效表达,且主要表达在血管区域。(2)HO-1在供肝的缺血再灌注损伤中对肝脏有显著的抗凋亡和抗炎的保护作用。并且抗凋亡作用与上调Bcl-2、Bcl-xl和下调caspase-3有关,而抗炎作用则可能与抑制巨噬细胞浸润和减少TNF-α的表达有关。更多还原

【Abstract】 Background:IRI (ischemia-reperfusion injury) is inescapable to liver transplantation, and may bring grafts on primary nonfunction which is one of the most important factors of retransplantation. So the study on alleviating IRI conduces to shrink the gap between organs available for transplantation and the number of patients awaiting an organ. Meanwhile, the gene therapy is one of the clinic ways to be practiced rapidly at recent years. It is an alluring therapeutic approach to utilize the technique of transforming functional genes into organ grafts to alleviate IRI.Part one:An Animal Model of ROLT by Modifying Kamada’s MethodObjective: to establish an animal model of ROLT by modifying Kamada’s method.Methods: 120 cases of ROLT were performed by ameliorating the surgical technique based on Kamada’s method and improving the treatment at around-operation period, and were investigated the operation time of donors and recipients, anhepatic period and survival time.Results: In contrast with the initial stage, there were significant differences from the operation time, the livability of 24 hours and one week and the incidence of the operation complication.Conclusion: It can establish an ideal animal model of ROLT via improving the surgical technique based on Kamada’s method and performing a certain amount of cases. Part two:Construction of Recombinant Adenovirus of Human Heme Oxygenase-1 GeneObjective: To construct the recombinant adenovirus of human heme oxygenase-1 gene.Methods: (1) The segment of hHO-1 gene was liberated from the cloning vector of pOTB7-hHO-1 by restriction endomuclease EcoRI and Bg1II; (2) the segment of hHO-1 gene was ligated into adenovirus shuttle vector pSGCMV after digested with EcoRI and Bg1II by standard procedure, and the production pSGCMV- hHO-1 was identified and selected. (3) pSGCMV- hHO-1 and backbone vector pBHGloxP△E1E3 were cotransformed into the adenoviral packaging 293 cells by the Cre-mediated sit-specific recombination method, and it was named Ad-HO-1. (4) After the security of Ad-HO-1 was evaluated by VT001/VT002, his infectional efficiency in vitro was also tested.Results: (1) the positive recombination pSGCMV- hHO-1 was obtained after hHO-1 gene was ligated into pSGCMV, and the hHO-1 gene of the verified pSGCMV- hHO-1 was correct. (2) Ad-HO-1 which was obtained by the pathway of the Cre-mediated sit-specific recombination method was in high titer which reached 2.0×1010pfu. (3) The obtained Ad-HO-1 did not contain wide-type adenovirus, and could be transformed effectively in vitro.Conclusion: Ad-HO-1 which can express effectively and safely in vitro can be obtained by the Cre-mediated sit-specific recombination method.Part three:hHO-1 gene transfer protects rat grafts from IRI followed OLTObjective: To investigate how recombinant adenovirus carrying HO-1 which was constructed by ourselves alleviate hepatic IRI after transplantation.Methods: Two groups of SD rats (n=8) were studied. Control donors were intraperitoneally pretreated with Ad-EGFP (2.5×109pfu) 36 hours before their livers were harvested; and experimental donors were intraperitoneally pretreated with Ad-HO-1(2.5×109pfu) 36 hours before their livers were harvested. 6 hours after reperfusion, serum ALT, AST and LDH were measured, and apoptotic cells by TUNEL and the apoptotic ratio by Flow Cytometer were analyzed; and the expression of HO-1 and antiapoptotic(Bcl-2 and Bcl-xl) and proapototic(caspase-3) gene products were determined by Western-blot. The positive cells of HO-1, TNF-α, caspase-3 and macrophage were stained by immunohistochemistry. The ultrastructure of grafts was observed by electronic microscope.Results: (1) The expression level of HO-1 in the experimental group was significantly higher than one in the control. Whereas the liver function indicator ALT, AST and LDH and the apoptotic ratio of hepatic cell decreased significantly. (2) By Western-blot,the expression of antiapoptotic(Bcl-2 and Bcl-xl) gene product in the experimental group was increased significantly, and the expression of proapoptotic(casepase-3) gene product was weakened obviously. (3)By immunohistochemistry ,as compared with the control group,in the experimental group a lot of the positive cells of HO-1 were expressed and mainly lay at the vascular area,and there were neither massive macrophages infiltration nor caspase-3 and TNF-αoverexpression. And there were more apoptotic cells around the Disse space in the control group than in the experimental group by TUNEL(.4)By electronic microscope,there were a few classic apoptotic cells to be observed in the control group,but the ultrastructure of the experimental group was almost normal.Conclusion: (1)The recombinant Ad-HO-1 can be intraperitoneally transfered into donor liver and be expressed obviously in vascular area of grafts. (2) HO-1 can alleviate ischemia-reperfusion injury in rat liver by suppressing apoptosis and inflammation which relates to significantly modulating proapoptotic (caspase-3) and antiapoptotic (Bcl-2 and Bcl-xl) pathways and depressing macrophage infiltration and TNF-αexpression.更多还原