【作者】 蒋国平；

【导师】 郑树森；

【作者基本信息】 浙江大学 ， 外科学， 2003， 硕士

【摘要】 目的 本实验利用封闭群SD大鼠到近交系Wistar大鼠的同种异体原位肝脏移植模型，以V型复制缺陷型腺病毒为载体，将CTLA4-Ig基因导入供肝内，以编码β-半乳糖苷酶的腺病毒（Adex/LacZ）为对照，探讨CTLA4-Ig基因对同种异体肝脏移植后的免疫耐受的诱导作用及机理：并通过体外实验研究重组CTLA4-Ig融合蛋白对巨噬细胞功能的影响。 方法 1．建立供体封闭群SD大鼠到受体近交系Wistar大鼠的同种异体原位肝脏移植模型。 2．分组：Ⅰ组（同基因移植组）为Wistar大鼠到Wistar大鼠肝移植；Ⅱ组（排斥对照组）为SD大鼠到Wistar大鼠肝移植；Ⅲ组（CsA处理组）为SD大鼠到Wistar大鼠肝移植后用环孢霉素A（CsA）3.0mg/kg/d肌肉注射，共注射13天；Ⅳ组（CTLA4-Ig基因组）为SD大鼠到Wistar大鼠肝移植前7天供体SD大鼠阴茎背静脉注射含有CTLA4-Ig基因的重组腺病毒液1×10⁹PFU，共一次；V组（LacZ⁺基因组）为SD大鼠到Wistar大鼠肝移植前7天供体SD大鼠阴茎背静脉注射含有LacZ⁺基因的腺病毒液1×10⁹PFU，共一次。 3．观察指标：各组大鼠的术后情况、生存时间。在移植后第1、3、5、7、12天时间段各杀死3只，酶链免疫吸附试验（ELISA）检测循环血中IL-2、IL-4、IL-10和IFN-γ水平；逆转录多聚酶链反应（RT-PCR）检测移植物中细胞因子表达；免疫组织化学（IHC）检测移植物中CTLA4-Ig基因的表达及巨噬细胞和CD₈⁺T细胞等免疫细胞浸润状况；TdT介导的dUTP缺口末端标记技术（TUNEL）观察移植物细胞凋亡情况；同时各时间段移植物的组织切片行排斥反应的病理学分级。 浙江大学硕十学位论文4．体外实验及观察指标：分离培养健康志愿者外周血的巨噬细胞，用含或不含LPS （10n／ml）的有血清培养基培养 6 ，J’时后，用 CsA 0．spg／ml、不同浓度的 CTLA4－Ig重 组蛋白及两者联合使用，分别处理巨噬细胞，并设相应浓度人Ig为对照，24小时后用 ELISA法检测细胞上清液中TGF－61、VEGF等细胞因子的表达。 结 果1．移植大鼠存活时间：排斥对照组移植大鼠存活时间平均为13．17天，与LacZ”基因组相近 （平均为12．33天）；与这两组相比，CTLA4｛g基因组和CsA处理组移植大鼠存活时间 明显延长 1’ )，差异有显著性意义(P（0．of）。2．CTLA4－Ig基因：静脉注射CTLA4－Ig基因重组腺病毒后7天的SD大鼠肝脏中己有CTLA4－Ig 基因表达，而未经处理的供体SD大鼠肝脏和阴茎背静脉注射含有LacZ基因的重组腺病 毒后7天的SD大鼠肝脏均无CTLA4－Ig基因阳性表达。CTL＾4－Ig组在移植术后60天， 移植物中仍能检测到CTLA4－Ig基因的表达。3．细胞因子变化：在排斥对照组和LacZ”基因组循环血中IL－2、IFN－v的表达水平相对较 高，而CTLA4－Ig基因组和CSA处理组IL－10的表达水平较前两组高。CSA处理组循环血 中细胞因子水平的变化介于排斥对照组与CTLA4｛ 基因组之间。4．免疫细胞浸润状况：排斥对照组和LacZ“基因组移植肝在术后第3天后CD。“T细胞浸润增 加；CTLA4｛g基因组的巨噬细胞浸润情况明显较其他组少；CSA处理组的巨噬细胞浸润 介于上述三组之间。CDS1细胞浸润方面，排斥对照组较CTLA4－Is基因组和CsA处理组 明显，CTLA4《g基因组＊1细胞浸润较CSA处理组轻。5，细胞凋亡：排斥对照组和LacZ“基因组凋亡指数（AI）明显高于CTLA4－Ig基因组和CSA 处理组，差异有显著性意义。CTLA4－IS组与CsA处理组相比较，各时间段差异无显著性。 在各级排斥反应中均可见凋亡细胞存在，发生凋亡的细胞主要是肝细胞，且凋亡指数（AI） 与急性排斥反应的分级成正相关。6．移植物病理排斥分级：在移植术后第3天及以后各时间段yLA4－Ig组和CsA处理组大鼠 的排斥程度明显比排斥对照组和L：。z组轻：第12天时间段CTLA4－比处理组大鼠的排斥 －2－ 浙江大学硕士学位论文 程度较CSA处理组轻。7．体外实验：经LPS刺激后的巨噬细胞在CTLA4－Ig融合蛋白处理组上清液中TGF BI含量 较低，并随着CTLA4｛g用药剂量的增加，TGF－el含量呈现下降趋势，而CSA处理组 TGF－pl含量明显增高，两者同时存在时，上清液中TGF－pl含量与CTLA4｛g蛋白单独 用药时的水平相近。在VEGF方面，CTLA4｛g蛋白组巨噬细胞上清液中VEGF的含量增高， 井丫剂量依赖性，而当CSA同时存在时，VEGF的含量下降。 结 论1．以腺病毒为载体的CTLA4刁g基因经静脉注射可以在大鼠肝脏内长时间稳定表达。2．腺病毒介导的 CTLA4｛ g基因可以诱导移植后受体细胞因子网络发生 Th—Th。转变， 并抑制移植物中免疫活性细胞的浸润和移植物细胞凋亡，从而诱导移植后免疫耐受， 延长受体生存时间，效果优于CSA。3．CTLA4－Ig通过与巨噬细胞表面B7分子结合而抑制巨噬细胞的趋化能更多还原

【Abstract】 ObjectivesThe objectives of the study were to explore the role of blocking B7/CD28 costimulation pathway with CTLA4-Ig gene in inducing tolerance after liver transplantation. Its relative mechanism was discussed too. Rat orthotopic liver allograft model was employed in this study. CTLA4-Ig gene was transducted into allograft with aid of adenovirus encoding P -lactase and serving as control in this study. The effect of recombinated CTLA4-Ig fussion protein on the function of macrophages was also studied in vitro in this research.Methods1. Establishing orthotopic liver transplantation model using SD rats to Wistar rats.2. Grouping: Group I (syngeneic control): Wistar to Wistar; GroupII (rejection control): SD to Wistar; Group IIKCsA-treated group): SD to Wistar recipients were treated with Cyclosporine A 3. Omg. kg"’, d"1 intramuscllarly after allografting from day 0 to day 12 postopration. Group IV (CTLA4-Ig-transducted group): SD to Wistar, the donor was pretreated with CTLA4-Ig-transducted recombinated adenovirus at dosage of 1X 109PFU, once 7 day before transplantation; Group V(LacZ+control):SD to Wistar, the donor was pretreated with LacZ+ gene-transducted adenovirus, by method as fore mentioned.3. Parameters: general situation and survival of each group were observed on 1,3,5,7, 12d. 3 rats were sacrificed. Blood samples were harvested for detecting of IL-2, IL-4, IL-10 and IFN-y by ELISA. Expression of cytokine in grafts was measured by reverse-transcription polymerase chain reaction. Respectively, the expression of CTLA4-Ig gene and the infiltrate of macrophage and CD8T cell in grafts were detected by immunolhistochemistry stainning. Cell apoptosis in grafts was detected by TUNEL. Morphologic changes of graft and severity of rejection were assessed too.4. Studies in vitro: the macrophages were isolated from peripheral blood of volunteers and stimulated with or without lOng/ml LPS for 6 hours, then the macrophages were treated with CsA(500ng/ml) and recombinated CTLA4-Ig fussion protein at various concentration . Expression of cytokines such as TGF-01, VEGF, IL-8 was observed after 24h.Results1. Survival: the average survival of rejection group is 13. 17 days, which is no difference with survival of LacZ+ control group (12. 33 days).In comparison to it, survival of recipients in CSA-treaed group and CTLA4~Ig-transducted group are statistically prolonged (P<0.01)2. Expression of cytokines: IL-2 and IFN-y were highly expressed in rejection group as well as in LacZ group; expression of IL-10 in CTLA4-Ig-transducted group and CsA-treated group is higher than that of rejection and LacZ group. The cytokine level in serum of CsA treated group was in the middle between theose of rejection andCTLA4-Ig-transducted group.3. Infiltration of immune cells: infiltration of CD87 cell are marked in rejection group and LacZ group 3rd post operation, whereas CD8*T cell infiltrition in CTLA4-Ig-transducted group was less than that in CsA-treated group. Infiltration of macrophage was significantly faint in CTLA4-Ig-transducted group than in other groups.4. Cell apoptosis: the apoptotic index of rejection and LacZ group was significantly higher than those of CTLA4-Ig-transducted and CsA-treated group, whereas there was no difference between CTLA4-Ig-transducted and CsA-treated group. Apoptotic cells were able to be observed in any rejection grades, the mainly cell of apoptosis in every group were hepatocyte.A good positive correlation was shown between AI and acute rejection.5. Histological assessment: the severity of rejection reaction after day 3 was weaker in CTLA4~Ig-Transducted group and CsA-treated group than in rejection control and LacZ control group, on day 12 after transplantation, severity of rejection was lower in CTLA4-Ig-transducted group than in CsA-treated group.6. In vitro study: TGF-31 expressed by LPS-stimulated macrophages was decreased after CTLA4-Ig fussion protein treatment and was in the dose dependent manner. TGF-p 1 was increased af更多还原