【作者】 陈志红；

【导师】 钱海鑫；

【作者基本信息】 苏州大学 ， 普通外科学， 2009， 博士

【摘要】 目的（1）改良二袖套法大鼠原位肝脏移植动物模型的成功建立。（2）改良二袖套法大鼠原位肝脏移植急性排斥动物模型的建立。（3）重组腺病毒pAdeasy-1-pAdTrack-CMV-OX40Ig（AdOX40Ig）的构建（4）研究重组腺病毒AdOX40Ig不同感染剂量对人肝细胞HL-7702生长的影响及其感染效率,筛选最佳感染剂量进行该重组腺病毒的生物学功能研究。（5）探讨腺病毒介导OX40Ig基因转移在诱导大鼠肝脏移植物长期存活中的作用和机制。方法（1）通过改良的双袖套法对130对SD大鼠进行了原位肝移植。前面40对为预试验组,中间60对为实验熟练组,后面30对为实验完善组。观察各组的手术成功率和术后生存率以及手术并发症。（2）采用改良二袖套法行Lewis大鼠到Brown Norway（BN）大鼠原位肝脏移植共30例,将其随机分成实验组15例,术后未进行特殊的干预,和对照组15例,术后1～7天用FK506 0.2mg/（kg.d）灌胃。术后4、7和10天的时间点上各组均随机处死3只受体大鼠,收集血清与肝脏标本,其余受体大鼠的血清标本通过舌静脉取血获得。血清标本检测细胞因子和生化指标,肝脏标本观察病理变化和肝细胞凋亡。每组各留6只观察术后生存情况及生存时间。（3）将经过双酶切的hOX40片段和hIgG1 Fc片段与同样经过双酶切的pAdTrack-CMV载体进行连接反应,并在大肠杆菌感受态细胞里转化成pAdTrack-CMV-hOX40Ig转移质粒,再通过PCR扩增双酶切及DNA测序鉴定,经鉴定正确的质粒命名为pAdTrack-CMV-OX40Ig（pTOX40Ig）。将pTOX40Ig转移质粒经PmeⅠ单酶切线性化后,与pAdeasy-1腺病毒载体氯化钙法共转化BJ5183感受态细胞,涂布卡那平板,挑选克隆、扩增,抽提质粒行PCR及PacⅠ酶切鉴定。鉴定正确的重组腺病毒质粒pAdOX40Ig经PacⅠ酶切后,采用Lipofectamine2000脂质体转染法转染293A细胞,转染第3天在荧光显微镜下观察荧光。重组腺病毒AdOX40Ig通过Western印迹鉴定。（4）将AdvOX40Ig重组腺病毒以1、10、25、50、100、200MOI不同剂量感染HL-7702细胞。在普通光镜视野下观察被感染后HL-7702细胞形态,在荧光视野下观察绿色荧光。将AdvOX40Ig重组腺病毒和Ad空载体腺病毒以50MOI剂量分别感染爬片的HL-7702细胞,用免疫荧光染色试剂盒进行间接免疫荧光检测腺病毒介导的外源性OX40Ig基因在HL-7702细胞中的表达;用ELISA法测定转染AdvOX40Ig的HL-7702肝细胞培养上清。（5）改良二袖套法行Lewis大鼠到Brown Norway（BN）大鼠原位肝脏移植40例,随机分成4组,每组10对。对照组:供、受体均未经任何处理,空载病毒AdEGFP处理组:术中供肝离体后肝内留存2ml的AdEGFP腺病毒[1×10⁹pfu/ml],AdOX40Ig处理组:术中供肝离体后肝内留存2ml的AdOX40Ig腺病毒[1×10⁹pfu/ml],FK506处理组:术后FK506(0.2mg·kg^-1·d^-1)灌胃。术后第7天每组各处死BN鼠3只,先经下腔静脉抽血,取部分血清,进行肝功能的检测,取肝脏,观察病理变化和肝细胞凋亡,取脾脏细胞与Lewis大鼠和第三品系F344大鼠的脾脏细胞进行单向混合淋巴细胞反应（MLR）。在BN大鼠与Lewis大鼠的MLR反应体系中,另取3个复孔,在每个复孔中加入20μl（1 IU/μl）重组IL₂。取四组处死BN大鼠的剩余血清及术前舌静脉采血取得的血清,采用双抗体夹心ABC-ELISA法来检测IFN-γ、IL-2、IL-4和IL-10的含量。余大鼠观察生存期,并且分别于病毒给予当天和第1、3、7、10、14、21和28天取大鼠尾静脉血,收集血清。用ELISA法检测血清中OX40Ig蛋白表达水平。结果（1）预试验组的手术成功率和7天生存率分别为27.5%和0;实验熟练组分别为75.0%和40.0%;实验完善组分别为93.3%和86.7%。（2）手术的成功率是93.7%（30/32）。实验组大鼠在术后7～10天表现出明显的急性排斥症状。实验组的生存时间是9.17±1.17（天）,而对照组是50.17±8.50（天）。实验组的肝功能（AST、ALT、TB）明显高于对照组。实验组术后第7天的血清IFN-γ和IL-2浓度明显高于术前,而IL-4和IL-10浓度则明显低于术前;对照组正好与此相反,术后第7天的血清IFN-γ和IL-2浓度明显低于术前,而IL-4和IL-10浓度明显高于术前。受体大鼠术后肝脏病理组织学检查发现,实验组术后第7天出现中度细胞性排斥,10天出现重度细胞性排斥,而对照组术后第7和第10天呈轻微细胞性排斥。此外,实验组肝细胞凋亡数较对照组显著增多。（3）经酶切反应及DNA测序鉴定,重组腺病毒质粒pAdOX40Ig序列正确。重组腺病毒质粒pAdOX40Ig转染293A细胞,转染第3天在荧光显微镜下能观察到荧光。反复冻融的重组病毒子经多轮感染后,检测效价达到10¹⁰pfu/ml。重组腺病毒AdOX40Ig的Western印迹鉴定可以看到48.7kD的条带。（4）在普通光镜视野下观察1、10、25、50、100MOI不同剂量重组腺病毒感染组HL-7702细胞均形态正常,生长良好;而200MOI剂量感染组细胞圆缩、脱落,呈现明显的细胞毒性;在荧光视野下,10、25、50、100、200MOI不同剂量重组腺病毒感染组HL-7702细胞几乎所有细胞均可观察到绿色荧光。AdvOX40Ig感染组HL-7702细胞能检测到Cy3红色荧光,而Ad空病毒感染组和未感染组HL-7702细胞则未出现红色荧光。ELISA测定显示,AdOX40Ig感染组细胞培养上清中OX40Ig浓度明显高于无病毒感染组和AdEGFP感染组。（5）AdOX40Ig处理组和FK506处理组受体大鼠存活时间明显超过对照组和空载病毒AdEGFP处理组,肝功能（AST、ALT、TB）明显低于对照组和空载病毒AdEGFP处理组,肝细胞性排斥明显低于对照组和空载病毒AdEGFP处理组,肝细胞凋亡也明显少于对照组和空载病毒AdEGFP处理组。术后AdOX40Ig处理组和FK506处理组受体大鼠血清中IFN-γ、IL-2的含量较术前明显降低（P＜0.05）,而IL-4和IL-10的含量则较术前明显升高（P＜0.05）;对照组和空载病毒AdEGFP处理组大鼠肝移植术后排斥组血清中IFN-γ、IL-2的含量较术前明显升高（P＜0.05）,而IL-4和IL-10的含量较术前明显降低（P＜0.05）。AdOX40Ig处理组和FK506处理组的CPM值均明显高于对照组和空载病毒AdEGFP处理组。在BN大鼠与Lewis大鼠的MLR反应体系中,加入重组IL-2,AdOX40Ig处理组和FK506处理组CPM值下降轻,与未加入重组IL-2的CPM值相比,差异有统计学意义。在BN大鼠与F344大鼠的MLR反应体系中,CPM值结果显示对照组、空载病毒AdEGFP处理组和AdOX40Ig处理组相互之间无统计学意义上的差异,但是这三组的CPM值均明显高于FK506处理组。结论（1）熟练和完善的外科操作技术是手术成功的关键;缩短无肝期时间是术后长期生存的有效保障。（2）以近交系Lewis大鼠为供体、BN大鼠为受体的组合发生的急性排斥反应强烈而且稳定,是理想的大鼠肝脏移植急性排斥模型。（3）重组腺病毒pAdOX40Ig构建成功,检测效价达到10¹⁰pfu/ml。（4）结果表明10、25、50、100MOI不同剂量病毒感染HL～7702细胞对细胞几乎无毒性,而且呈很高的感染效率,其可达90%以上,这提示10MOI为最佳感染剂量。间接免疫荧光检测结果和ELISA法测定结果均表明,腺病毒介导的外源性OX40基因能在HL-7702细胞中成功表达。（5）用OX40Ig处理供肝具有FK506的抗急性排斥反应的功能,具体表现在,用AdOX40Ig处理的大鼠存活时间长,肝细胞性排斥轻,肝细胞凋亡少,肝功能损害轻,免疫反应向Th2细胞偏移,被抑制的混合淋巴细胞反应能够被IL-2逆转。用AdOX40Ig处理供肝,能够抑制用同品系大鼠的刺激细胞引起的混合淋巴细胞反应,但不能抑制用异品系大鼠的刺激细胞引起的混合淋巴细胞反应,这与FK506不同。更多还原

【Abstract】 Objectives（1） To set up a successful model of orthotopic liver transplantation in rats by modified two-cuffed technique..（2） To set up an acute rejection model of orthotopic liver transplantation in rats by modified two-cuffed technique.（3） To construct a recombinant adenovirus vector pAdeasy-1-pAdTrack-CMV-OX40Ig （AdOX40Ig）.（4） To determine the influence and infection efficiencies of different titers of AdOX40Ig recombinant adenovirus on the growth of human hepatocyte HL-7702,and select out the best titer to study on the biological function of the virus.（5） To discuss the role and regime of adenovirus-mediated OX40Ig gene in inducing long-term survival of liver allografts in ratsMethods（1） Orthotopic liver transplantation was conducted in 130 pairs of SD rats by modified two-cuffed technique.The first 40 pairs are the pre-experimental group,the middle 60 pairs are the experimentally skilled group,and the last 30 pairs are called the experimentally consummate group.The successful operative rate,the postoperative survival rate and the operative complications were observed.（2） Thirty cases of Orthotopic liver transplantation was performed in Lewis to BN rats through the modified two-cuffed technique,and all rats were randomly divided into 2 equal groups.One was experimental group which were not specially treated,and the other was contrast group which took gastric perfusion with 0.2mg/（kg.d） FK506 during 1～7 days after operation.Each time on the 4th,5th and 10th days after transplantation,3 rats in either group were killed,whose blood and liver samples were collected while the other receptor rats’ blood samples were drawn from their lingual vein.Blood samples were used for examining cytokine and biochemical indicators.Liver lobes were applied to detect pathological changes and liver cell apoptosis.The 6 survival rats in each group were used for recording postoperative living status and survival time.（3） After double digestion,hOX40,hIgG1 Fc fragment and pAdTrack-CMV vector were connected for gene recombination,then transferred into competent E.coli cells and generated pAdTrack-CMV-hOX40Ig transfer plasmid.It was verified by PCR amplification,double-enzyme digestion and DNA sequencing,the correct plasmid named pAdTrack-CMV-OX40Ig（pTOX40Ig）.After linearization of transfer plasmid PTOX40Ig,the plasmid was co-infected with pAdeasy-1 adenovirus vector into competent BJ5183 cells by the method of calcium chloride.Then the cells were coated on the tablet containing kanamycin.Correct clone was selected by extraction of plasmid,and thereafter PCR and restriction enzyme digestion by PacⅠfor verification.The correct recombinant plasmid pAdOX40Ig was transfected into 293A cells by using Lipofectamine 2000 after digested by PacⅠ.On the third day after tansfection,the fluorescence was observed under fluorescence microscope.The recombinant adenovirus AdOX40Ig was also identified by Western blot.（4） Recombinant adenovirus AdvOX40Ig infected HL-7702 cells at different titer such as 1,10,25,50,100,200 MOI.Cell morphology of infected HL-7702 was observed under light microscope,and the green fluorescent was observed under the fluorescent vision. AdvOX40Ig recombinant adenovirus and mock adenovirus were infected into HL-7702 cells at 50MOI dose respectively.Adenovirus-mediated exogenous gene OX40Ig expression in HL-7702 cells was detected through indirect immunofluorescence by immunofluorescent staining kit.ELISA method was also used to determine the concentration in the culture supernatant after HL-7702 liver cells infected with adenovirus AdvOX40Ig.（5） Forty cases of Orthotopic liver transplantation was performed in Lewis to BN rats through the modified two-cuffed technique,and all rats were randomly divided into 4 equal groups:contrast group（neither donors nor receptors were treated）,AdEGFP treated group (donor liver carried 2ml adenovirus AdEGFP[1×10⁹pfu/ml]after it was excised during operation,AdOX40Ig treated group（donor liver carried 2ml adenovirus AdOX40Ig [1×10⁹pfu/ml]after it was excised during operation）,and FKS06 treated group(gastric perfusion with FK506(0.2mg·kg^-1·d^-1) after operation).On the 7th day after operation,3 BN rats in each group were killed.Blood was then drawn from the inferior caval vein to detect the liver funtion.Liver was excised to detect pathological changes and liver cell apoptosis.Spleen cells were collected to undergo mixed lymphocyte reaction（MLR） with the Lewis rats and the third strain F344 rats’ spleen cells. Three repeated holes were prepared in the MLR process between BN rats and Lewis rats among which each was mixed with 20μl（1 IU/μl） Recombinant IL-2.With the remaining blood of the 4-group killed BN rats and the blood preoperatively drawn from lingual vein, the content of IFN-γ,IL-2,IL-4 and IL-10 was detected by double antibody sandwiched ABC-ELISA procedure.The survival rats were used to detect live range,and their blood was collected from caudal vein on the day virus was planted and on the 1st,3rd,7th,10th, 14th,21th and 28th days after that treatment.The OX40Ig protein expression level was assayed ELISA procedure. Results（1） The successful operative rate and the 7-day survival rate were respectively 27.5% and 0 in the pre-experimental group,75.0%and 40.0%in the experimentally skilled group, and 93.3%and 86.7%in the experimentally consummate group.（2） The successful operative rate was 93.7%（30/32）.The experimental group of rats exhibited obvious acute rejection on the 7th～10th days after operation.The live range was 9.17±1.17 days for the experimental group and 50.17±8.50 days for the contrast group.The liver function of the experimental goup was obviously better than that of the control group. As for the experimental group,the content of IFN-γrand IL-2 on the 7th postoperative day was significantly more than that in the preoperative time,vice versa for IL-4 and IL-10.As for the contrast group,the result was opposite.The postoperative hepatic pathological results showed that medium and severe cellular rejection turned up respectively on the 7th and 10th postoperative days in the experimental group while in the contrast group mild cellular rejection turned up on the same days.The liver cell apoptosis was distinctively more severe in the experimental group than that in the contrast group.（3） By enzyme digestion and DNA sequencing,the sequence of recombinant adenovirus vector pAdOX40Ig was proved to be correct.In infection of 293A cells,could be seen on the third after transfection.After several rounds of infection of recombinant virus,testing titer reached 10¹⁰pfu/ml.A strip of 48.7kD could be seen from Western blot on identification of recombinant AdOX40Ig.（4） Under light microscope,recombinant adenovirus infected HL-7702 cells at 1,10,25,50,100 MOI of different doses were morphologically normal,grow well,and infected cells at 200MOI were round shrinkage and off shown clear signs of cell toxicity. At fluorescence Perspective,the green fluorescence were almost all could be observed in recombinant adenovirus infected HL-7702 cells at 10,25,50,100,200 MOI. AdvOX40Ig infected HL-7702 cells could be detected with the Cy3 red fluorescence, while the mock virus infection group and non-infection group HL-7702 cells couldn’t be seen in red fluorescence.Determination of ELISA results showed that,AdOX40Ig infection group was significantly higher than non-infected group and AdEGFP infected group.（5） The living range of rats in the AdOX40Ig and FK506 treated groups was obviously longer than that in the control and AdEGFP treated groups,while the hepatic function of the previous groups was worse than that of the latter groups,the previous’ hepatic cellular rejection was obviously milder than the latter’s,and the previous’ liver cell apoptosis was less than the latter’s.The content of IFN-γand IL-2 in the blood after operation was obviously lower than that before operation（P＜0.05）,vice versa for the content of IL-4 and IL-10（P＜0.05） in the AdOX40Ig and FK506 treated groups.As for the contrast and AdEGFP treated grous,the the postoperative content of IFN-γand IL-2 was obviously higher than before operation（P＜0.05） while the postoperative content of IL-4 and IL-10 was obviously lower than before operation P＜0.05).The CPM indexes of the AdOX40Ig and FK506 treated groups were obviously higher than that of the contrast and AdEGFP treated groups.With respect to the MLR reaction between BN rats and Lewis rats,the CPM indexes of AdOX40Ig and FK506 treated groups were significantly higher when the MLR was mixed with recombinant IL-2 than when the MLR was not mixed with recombinant IL-2.As for the MLR reaction between BN rats and F344 rats,there’s statistically no meaning in comparison between each other about the CPM indexes of contrast,AdEGFP and AdOX40Ig treated groups.Nevertheless,the CPM indexes of the three groups were significantly higher than the FK506 group.Conclusions （1） The skilled and consummate surgical operative technique is key to successful operations.Shortening the anhepatic period efficiently ensures the long-term survival after operation.（2） The acute rejection reaction between Lewis rats as donors and BN rats as receptors was fierce and stable,and thereof,an ideal acute rejection model for liver transplantation in rats.（3） The recombinant adenovirus pAdOX40Ig was successfully constructed and the titer of virus was 10¹⁰pfu/ml.（4） Our results showed that different titers of virus such as 10,25,50,100MOI were not only non-toxic to cells but with high infection efficiency ahnost up to 90%.All these showed that 10MOI maybe the best infection titer.The results of indirect immunofluorescence test and ELISA showed that adenovirus induced exogenous OX40 gene expressed successfully in HL-7702 cells.（5） The donor liver with OX40Ig treatment had the function of countering acute rejection,just like FK506.Specifically,the rats with AdOX40Ig treatment had a longer living range,milder hepatic cellular rejection,less hepatic cellular apoptosis,lighter harm to hepatic function,immunoreaction toward Th2 shift,and reversibility of depressed mixed lymphocyte reaction by IL-2.Unlike FK506,the donor liver with AdOX40Ig treatment could depress mixed lymphocyte reaction induced by activating cells of the same strain rats but could not depress mixed lymphocyte reaction induced by activating cells of rats of different stains.更多还原