【作者】 宇汝胜；

【导师】 钱海鑫；

【作者基本信息】 苏州大学 ， 外科学， 2008， 博士

【摘要】 背景:我国是世界上肝病发病率最高和因肝病导致死亡人数最多的国家之一,同时也是接受肝移植治疗的患者较多和肝移植发展较快的国家。肝移植后,肝脏无功能或肝衰仍然是一个重要的临床问题。同时,移植器官的短缺迫使人们不得不采用一些边缘供肝,而这些供肝对缺血再灌注损伤更敏感,使移植肝原发性功能障碍的发生率显著提高,这在相当程度上限制了肝移植的发展。如何避免这些损伤是目前肝移植研究领域的重点和热点。研究表明,肝移植缺血再灌注损伤过程中,NF-κB的激活导致其下游基因-炎性介质的大量表达,从而在移植后缺血再灌注损伤过程中发挥重要作用。因此,我们可以设想抑制NF-κB的激活或表达有望调节或减轻缺血再灌注损伤。RNAi技术可以高效沉默特定基因,去除移植过程中不利基因的表达,在器官移植方面具有广泛的应用前景。有效的基因治疗方案需要将目的基因稳定、高效地转入移植物的细胞。腺病毒载体因具有嗜肝性、能够转染处于非增殖期的实质细胞而在肝脏疾病研究方面具有优势。第一部分siRNA转染BRL细胞时转染条件的优化目的:探讨用RNAi-mate试剂转染siRNA至BRL细胞的最佳转染条件。方法:设立不同的2种RNAi-mate量和4种siRNA量,交叉组合成8个不同比例的复合物,即8个实验组,分别转染接种培养的BRL细胞,24小时后,观察细胞阳性转染率和细胞存活率。结果: siRNA的量为1.5ug,RNAi-mate为3.0ul时,其转染效率为89%,细胞的生存率为84%,优于其它条件时的转染效率并能保证相对小的细胞毒性。结论:在本实验中,用siRNA转染BRL细胞,当siRNA与RNAi-mate比例为1:2时,为最佳转染条件。第二部分siRNA对BRL大鼠肝细胞NF-κB p65表达影响的研究目的:筛选出能高效抑制NF-κB p65表达的siRNA序列。方法:设计制备4对针对大鼠NF-κB p65的siRNA,结合对照共分为7个实验组,采用研究一的转染条件体外转染培养的BRL细胞,采用逆转录PCR和Western blotting检测siRNA的基因沉默效果。结果:设计合成的4对siRNA均不同程度的从mRNA和蛋白表达水平抑制了BRL细胞NF-κB p65的表达,其中第二对siRNA mRNA和蛋白水平的抑制率分别为90%和80%。结论:4对siRNA转染BRL细胞后,均能发挥RNA干扰作用。第二对siRNA抑制效率最高。第三部分针对NF-κB p65的siRNA重组腺病毒的制备及病毒包装、扩增和滴度测定目的:构建针对NF-κB p65的siRNA重组腺病毒。方法:将NF-κB p65 shRNA表达框插到穿梭质粒pShuttle H1的启动子下游,鉴定包含目的基因的正确克隆,将其线性化后与腺病毒骨架质粒pAdEasy-1体外同源重组,共转化感受态细菌,筛选重组子并扩增。重组子线性化后,转染293A细胞中进行病毒的包装,同时大量扩增病毒,氯化铯密度梯度离心纯化。重组腺病毒转染BRL细胞检测NF-κB p65基因沉默效率。结果:穿梭载体插入序列及重组腺病毒质粒的鉴定均完全正确。病毒扩增纯化后滴度为3.0×1010 pfu/ml,并能在体外抑制NF-κB p65蛋白表达。结论:成功构建了针对NF-κB p65的siRNA重组腺病毒。第四部分大鼠原位肝脏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”的基础上,在取肝、肝上下腔静脉的吻合、围手术期处理等几方面进行改良,共施行大鼠原位肝移植动物实验140例,观察术后并发症及存活率。结果:经过140例次的训练,建立了稳定的大鼠原位肝脏移植模型。在实验的第三阶段,受体无肝期平均12分钟,手术成功率93%,两周存活率90%。结论:改良的二袖套法具有无肝期短、手术成功率高、大鼠术后存活时间长的优点,是大鼠原位肝移植的理想术式。娴熟细致的外科操作、受体无肝期的长短是决定动物存活的关键。第五部分Ad-shRNA-p65在大鼠移植肝缺血再灌注损伤中的保护作用目的:观测构建的Ad-shRNA-p65腺病毒载体在大鼠体内的基因沉默效果及其在大鼠原位移植肝缺血再灌注损伤中的保护作用。方法:术前36小时将Ad-shRNA-p65通过门静脉注入实验组供体大鼠肝脏,3.0×109/只。移植术前处死一批大鼠,检测血ALT、AST、LDH,肝脏病理,NF-κB p65的mRNA、蛋白表达。供肝获取后冷保存4小时,改良的二袖套法行原位肝脏移植术。门静脉复流6小时后观测受体胆汁分泌情况,处死受体大鼠,检测血ALT、AST、LDH,肝脏病理,肝组织MDA水平,NF-κB p65的mRNA、蛋白表达以及免疫组化;TNF-α、MIP-2、ICAM-1mRNA表达,TNF-α的免疫组化,TUNEL法检测肝脏细胞凋亡水平。结果:移植术前,实验组NF-κB p65的mRNA、蛋白表达降低,实验组和空病毒组血生化指标升高。术后6小时,实验组胆汁分泌量多于对照组,血生化指标降低,MDA水平、NF-κB p65的mRNA、蛋白表达明显低于对照组,TNF-α、MIP-2、ICAM-1mRNA低表达,实验组细胞形态变化较轻,凋亡水平明显低于对照组。结论:构建的腺病毒载体Ad-shRNA-p65能成功地转染供肝,并有效沉默NF-κB p65表达,转染本身对供肝可能有轻微的损害。Ad-shRNA-p65预处理的供肝能够抑制移植肝缺血再灌注损伤引起的NF-κB p65激活和表达,并减轻移植肝的缺血再灌注损伤,其机制与下调TNF-α、MIP-2及ICAM-1表达,进而减少肝脏内中性粒细胞的浸润有关。更多还原

【Abstract】 Background: Our country is one of countries with the higher morbidity and mortality of liver ailment in the world. Liver transplantation is developing quickly and the number of patients been performed liver transplantation is increasing in our country. Liver dysfunction or failure is a significant clinical problem after liver transplantation. The dramatic organ shortage forces lots of marginal grafts been used. These grafts have a higher susceptibility to ischemia-reperfusion injury and a higher possibility of primarily liver dysfunction after transplantation, which directly limits the developing of clinic liver transplantation. How to avoids or limits its happen is the focal point of the liver transplantation research.Some research indicated, in the processes of the ischemia-reperfusion injury of liver transplantation, the activation of NF-κB which leading into the expression of lots of inflammation cytokines plays a very important role. So we can suppose inhibit the activation or expression of NF-κB maybe have a use to lessen the ischemia-reperfusion injury in these process. The technique of RNA interference has a dominantly use of silencing special gene expression and an extensively utilize prospect in organ transplantation. Effective gene therapy requires a reliable gene transfer tools to efficiently insert target genes into the cells of the grafts. Adenovirus vectors have been favored because of their propensity to infect hepatic cells and their ability to infect proliferation and nonproliferation cells.PART I:Optimize the parameters for transfecting siRNA into cultivated rat BRL cells.Objective: To optimize the parameters for transfecting siRNA into cultivated rat BRL cells by RNAi-mate in vitro.Methods: Two different density of RNAi-mate were mixed with four different density of fluorescin - labeled siRNA respectively, and then eight mixtures were got. Each of the mixtures was used to transfect BRL cells.24 hours after transfection; cell count was made by fluorescent microscope to determine the percentage of the cells been transfected under each condition. The cell viability was calculated by using MTT assay at the same time.Results: In the case of 1.5ug siRNA plused with 3.0ul RNAi-mate, we found the transfection efficiency was 89%, the cell survival rate was 84%, and it was the best parameter.Conclusions: In our experiment, when transfectting BRL cells, the best ratio of siRNA: RNAi-mate was 1:2.PART II: The gene silence effect of the siRNA on the NF-κB p65 expression of the BRL cellsObjective: To choose the NF-κB p65 siRNA with the highest gene silence efficacy from the 4 pairs of siRNA.Methods: We designed 4 pairs of siRNA targeting rat NF-κB p65 mRNA. Combined with control group there were 7 experiment groups altogether. Then we transfected those siRNA into the cultivated rat BRL cells use the way we conformed in part 1. The NF-κB p65 gene expression effectiveness was tested by reverse transcription-PCR and Western blotting.Results: Though the suppressing degree was different, each one of those 4 pairs of siRNA could suppress the NF-κB p65 gene expression from the mRNA and protein level. The second siRNA was the most efficient one and the NF-κB p65 mRNA expression was decreased 90%, the protein expression was decreased 80% compared with the blank group.Conclusions: Our experiment confirmed, each one of those 4 pairs of siRNA has the function of gene silence and the second one was the most efficient one.PART III: Construct a recombinant Ad containing NF-κB p65 specific siRNA expression cassette and its amplification, purifying and titrating.Objective:To construct a recombinant Ad containing NF-κB p65 specific siRNA expression cassette.Methods:The NF-κB p65 specific siRNA expression cassette was cloned behind the sequence of H1-RNA promoter, and the positive clones containing target gene were selected and appraised. A recombinant adenovirus was produced by a double-recombination event between cotransformed adenoviral backbone plasmid pAdEasy-1 and a linearized shuttle vector. Positive clones were selected and confirmed. Then they were linearized and transferred into the 293A cell to amplification, and then were purified by density gradient ultracentrifuge and titrated. The desired Ad vectors were transfected into the BRL cells to detect the gene silence efficiency.Results:Both the plasmid pShuttle-H1-p65 and the recombinant Ad were confirmed correct. The recombinant Ad were generated in a high titer which reached 3.0×1010 pfu/ml, and could efficiently knock down the NF-κB p65 expression in vitro.Conclusion: we successfully constructed the recombinant Ad which containing NF-κB p65 specific siRNA expression cassette.PART IV: The establishment of a model of rat orthotopic liver transplantationObjective:To establish a reliable animal model of rat orthotopic liver transplantation with modified cuff technique.Methods:140 cases of rat orthotopic liver transplantation were performed by using the two-cuffed technique with some modification in graft harvesting, the anastomoses of suprahepatic vena cava and the perioperative treatment. Results:Of the last 30 cases, the mean time of anhepatic period was 12min, The 24 hours and 2 week survival rates were 93% and 90% respectively.Conclusions: The modified two-cuffed technique is convenient and easy to repeat. It can enhance the stability and survival rate of rat orthotopic liver transplantation models. Skilled surgical techniques and shortening the anhepatic phase of recipient as much as possible are the keys to animal survival.PART V: Adenovirus expressing shRNA targeting p65 have the protective effect against the ischemia-reperfusion injury of rat liver graftsObjective:To test the Gene silencing efficacy of Ad-shRNA-p65 in vivo and its potential protective effect against the ischemia-reperfusion injury of liver grafts.Methods: The donor rats was infused with the Ad- shRNA- p65 in a titer of 3×109 pfu via portal vein. 36 hours later, half of the rats were executed to detect the items as below: Serum ALT、AST、LDH; the liver pathology changes; the expression of NF-κB p65 mRNA and protein in the liver. The other donor rats were re-laparotomized to harvest the grafts and the grafts were preserved for 4 hours in Ringer’s lactate solution. Then the orthotopic rat liver transplantation (OLT) was performed using the modified two-cuffed technique described in the partⅣ. Six hours after transplantation, the expression of NF-κB p65 mRNA and protein and TNF-α、MIP-2、ICAM-1mRNA in the liver were tested; the bile volume, content of MDA in the liver and serum ALT、AST、LDH were measured, while NF-κB p65 and TNF-αcontent were estimated by immunohistochemistry and apoptosis was analyzed by TUNEL.Results:The mRNA and protein expression of NF-κB p65 were decreased in the donor rats which infused in Ad- shRNA- p65. and infusing Ad would induce the heighten of serum ALT、AST、LDH. Six hours after reperfusion, in the experiment group, the bile volume was increased, the expression of NF-κB p65 mRNA and protein , TNF-α、MIP-2、ICAM-1mRNA in the liver, the content of MDA in the live, the level of ALT、AST、LDH in serum were all decreased, compared with control groups. The immunohistochemistry showed that lower expression of NF-κB p65 and TNF-αin the liver tissue of the experiment group and TUNEL showed less apoptosis cells .Conclusions: Administered via portal vein, the recombinant adenovirus expressing shRNA targeting p65 could obviously silencing the NF-κB p65 expression in rat liver. Infuse in Ad itself maybe had a little degree of liver damage. Donor adenovirus-mediated gene transfer of Ad- shRNA- p65 could decrease subsequent ischemia-reperfusion injury in rat liver transplantation. One of its mechanism maybe explained as down-expression of NF-κB p65 will lead into the inhibit expression of TNF-α、MIP-2、ICAM-1, and thus reduce the leukocytes infiltrating the liver.更多还原