【作者】 江雯；

【导师】 魏泓；

【作者基本信息】 第三军医大学 ， 动物学， 2012， 博士

【摘要】 一、背景CTLA-4Ig(human cytotoxic T-lymphocyte-associated antigen4-immunoglobulin)是CTLA-4分子的细胞外功能区与人免疫球蛋白IgG1恒定区相结合的融合蛋白。它是功能明确、经典的免疫抑制分子，可与B7结合，阻断CD28/B7正性共刺激信号通路，诱导T细胞免疫无反应。许多研究都证实了其可以延长移植物存活时间。本实验室前期与西南医院烧伤研究所吴军教授课题组合作，利用hCTLA-4Ig基因重组腺病毒体外感染非转基因猪皮肤，成功研制了离体基因修饰活性皮肤[1]，也有效延长了活性猪皮肤在人体创面的存活时间。然而，全身性表达CTLA-4Ig的转基因动物难以成活。研究报道敲除CTLA-4基因的小鼠及全身高表达pCTLA-4Ig的转基因猪都在出生后不久死亡，提示CTLA-4Ig对转基因动物自身免疫功能有一定的影响，若调控性、限制性的表达CTLA-4Ig可能会降低该蛋白对转基因动物全身免疫功能的影响，提高制备转基因动物的成功率。大面积烧伤病人由于自体及异体皮肤不足以满足移植需要，猪皮肤作为临时替代和覆盖物已在临床上应用。皮肤是非血管化器官，其移植后的排斥反应主要以细胞排斥为主。根据本实验室前期的实验，以hCTLA-4Ig腺病毒体外修饰的基因转染猪皮能够显著延长其猪皮在烧伤病人创面存活时间，但体外病毒转染方法为单次转染单次应用，涉及病毒制备及转染工艺，成本高、工艺复杂、效率低、稳定性差、质量控制难度高。因此，构建皮肤靶向表达hCTLA-4Ig并可稳定遗传的转基因猪群体或品系作为皮肤供体，可望形成稳定、高效、经济的异种移植皮肤来源。巴马香猪是我国小型猪群体中唯一具有白色被毛的品系，且体表白毛面积大、繁殖力强、抗病力强、性格温驯，生物学特性明确，是适合的医学实验动物。利用标准化的巴马香猪构建转基因猪种群，可望提高转基因猪品系或群体的标准化、提高供体猪皮肤一致性，从而提高模型研究科学性、产品稳定性。研制皮肤靶向表达hCTLA-4Ig转基因巴马香猪是本实验研究最终目的。但由于构建转基因猪周期长、成本高、风险大，而小鼠繁殖力强、世代间隔短、研究时间短、经济成本低，因此在构建转基因猪以前构建转基因小鼠有利于提高研究的可控性。同时，构建小鼠、猪的皮肤靶向表达hCTLA-4Ig转基因动物模型，形成从小动物到大动物的模型体系，更有助于深入、系统研究hCTLA-4Ig及其在异种移植中的作用及机理。二、目的（一）建立小鼠、猪的皮肤靶向表达hCTLA-4Ig转基因动物模型，为系统研究hCTLA-4Ig分子对异种皮肤移植排斥反应的调控功能，提供从小动物（小鼠）到大动物（猪）的模型体系。（二）建立皮肤靶向表达hCTLA-4Ig转基因巴马香猪群体，为研制高效、稳定、经济的异种移植皮肤产品形成基础。三、方法（一）皮肤靶向表达hCTLA-4Ig转基因小鼠制备、种群建立及其功能验证通过慢病毒载体的方法构建皮肤靶向表达hCTLA-4Ig转基因小鼠，并建立封闭繁育群体；用PCR方法进行阳性小鼠的筛选和鉴定；用RT-PCR、real time PCR、ELISA、WB和免疫组化的方法研究转基因蛋白在小鼠体内的表达和分布；用单向MLC的方法，研究该转基因蛋白在体外的生物活性；用小鼠→大鼠异种皮肤移植实验模型，观察移植皮肤的存活状况、时间，研究该转基因蛋白在体内的生物学活性。（二）皮肤靶向表达hCTLA-4Ig转基因巴马香猪的构建以体细胞核移植方法构建皮肤靶向表达hCTLA-4Ig转基因巴马香猪；以PCR方法进行转基因阳性猪筛选和鉴定；以WB方法初步分析转基因猪体内该蛋白的表达和分布。（三）皮肤靶向表达hCTLA-4Ig转基因巴马香猪种群建立及其异种移植研究通过体细胞核移植的方法构建皮肤靶向表达hCTLA-4Ig转基因巴马香猪，并形成封闭繁育群体；用PCR方法进行阳性转基因猪的筛选和鉴定；以ELISA和WB的方法研究转基因蛋白在猪体内的表达和分布；以单向MLC的方法，研究该转基因蛋白在体外的生物活性；以猪→大鼠异种皮肤移植实验模型，观察移植皮肤的存活状况、时间，研究该转基因蛋白在体内的生物学活性；以病理切片HE染色来观察皮肤移植后的组织学变化。四、结果（一）建立了皮肤靶向表达hCTLA-4Ig的转基因小鼠种群，并证实了该转基因蛋白在小鼠皮肤组织的特异性表达及其生物学活性。根据PCR结果证实本实验获得8只hCTLA-4Ig转基因首建小鼠，转基因首建鼠的得率（转基因首建小鼠数/注射转染的胚胎数）是6.3%；并且通过这8只转基因首建小鼠的横交繁殖、PCR筛选，建立了皮肤靶向表达hCTLA-4Ig转基因小鼠种群；RT-PCR和Western blot分析表明，hCTLA-4Ig蛋白实现了在转基因小鼠皮肤中的特异性表达，且表达水平较高；免疫组化分析也表明，hCTLA-4Ig在皮肤中的特异性表达，且该转基因蛋白的表达对皮肤组织的正常发育和组织形态没有明显影响；ELISA分析表明，hCTLA-4Ig蛋白因其呈可溶性表达，在转基因小鼠血清中也有明显的分布，其浓度达11.87μg/ml；在单向MLC实验中，小鼠血清中转基因表达的hCTLA-4Ig蛋白明显抑制了人淋巴细胞混合培养系统中反应细胞的增殖，其程度与相同终浓度的纯化重组蛋白hCTLA-4Ig相当；在小鼠→大鼠皮肤移植实验中，hCTLA-4Ig转基因小鼠的皮肤比相同品系野生型小鼠皮肤在受体的存活时间明显延长。（二）获得了皮肤靶向表达hCTLA-4Ig的转基因巴马香猪种群，并证实了该转基因蛋白在巴马香猪皮肤组织的特异性表达及其生物学活性。成功获得了皮肤靶向表达hCTLA-4Ig的转基因克隆巴马香猪，首建猪得率是0.75%；通过转基因首建巴马香猪与野生型巴马香猪繁殖获得的F1代进行横交繁殖，经PCR筛选，建立了皮肤靶向表达hCTLA-4Ig的转基因巴马香猪种群；对F1代转基因猪进行PCR鉴定，在出生的59头F1代猪中有32头阳性（54.2%）；ELISA分析表明，hCTLA-4Ig蛋白因其呈可溶性表达，在转基因巴马猪血清中也有明显的分布，随机选取的5头F1代转基因巴马香猪血清中hCTLA-4Ig蛋白的浓度在5.61～9.56μg/ml之间；Western blot分析表明，hCTLA-4Ig蛋白在转基因猪的皮肤、角膜都有表达，但其表达水平稍低于内参基因GAPDH，而野生型巴马香猪的皮肤没有明显的表达条带；在单向MLC实验中，猪血清中转基因表达的hCTLA-4Ig蛋白明显的抑制了人淋巴细胞混合培养系统中反应细胞的增殖，其程度与相同终浓度的纯化重组蛋白hCTLA-4Ig相当；在猪→大鼠皮肤移植实验中，hCTLA-4Ig转基因巴马香猪皮肤比野生型猪皮肤异种移植到大鼠背部后存活时间延长；组织石蜡切片HE染色结果发现，宿主对移植物的反应程度具有差异，对转基因猪皮肤的炎症反应明显低于对野生型猪皮肤。五、结论（一）成功建立了皮肤靶向表达hCTLA-4Ig的转基因小鼠和转基因巴马香猪种群，通过体内外实验证实了转基因小鼠和转基因巴马香猪体内表达的转基因蛋白hCTLA-4Ig的生物学活性，为深入、系统研究hCTLA-4Ig及其在异种移植中的作用及机理，形成了从小动物到大动物的模型体系。（二）建立了皮肤靶向表达hCTLA-4Ig转基因巴马香猪种群，其种群可望成为临床异种移植供体皮肤来源，为研制高效、稳定、经济的异种移植皮肤产品形成基础。更多还原

【Abstract】 1. BackgroudThe fusion protein CTLA-4Ig(Cytotoxic T-lymphocyte-associated antigen4-immunoglobulin),which combined the extracellular domain of CTLA-4with the Fc portion of IgG1, can bindwith B7, directly blocking CD28-B7co-stimulation pathway, inducing T cell anergy. Manystudies confirm that the protein can prolong the survival time of grafts. Our previous study, invitro, adenovirus-mediated CTLA-4Ig gene transfer prolongs pig skin xenotransplantsurvival.However, directly expressing CTLA-4Ig in all over transgenic animal body, will make ithard to get the alive transgenic animal. CTLA-4gene knockout mice and transgenic pigsexpressing porcine CTLA-4Ig are died soon after born, suggesting that CTLA-4Ig may affectthe immune system of the transgenic animal. Therefore, modulately and restrictly expressingCTLA-4Ig may decrease the influence, increase the efficiency of preparation of transgenicanimals.Xenogeneic skin is frequently used to cover large wounds as a substitute for human skin,so it has litte danger to use in clinic. Moreover, Skin as a non-vascular organ，thetransplantation rejection is mainly depending on cell-mediated rejection. Allthrough ourprevious study prolongs pig skin xenotransplant survival by adenovirus-mediated CTLA-4Iggene transfer, the procedure of the gene transfer is complicated, low efficiency, high cost andrelated to preparing virus. Therefore, establish a population of skin specifically expressionhuman CTLA-4Ig transgenic pig as donor supply for clinlic, will be a low cost, highefficiency, easy way to solve the problem.Bama miniature pig is the only strain who has white skin in all miniature pig lines ofchina., and it has large area of white skin, high fecundity, strong resistance to illness, docileand clear biological characteristics, so it is most suitable for medical experiment. Usingstandardized bama miniature pig to establish transgenic pig populatin will promote the standardization and consistency of doner skin, guarantee the preciseness of model scientificresearch and the stability of the product.Our ultimate goal of this study is establishing a population of skin specificallyexpressing human CTLA-4Ig transgenic pig, but it will take long time because of the longreproductive cycle of pig, and will take high cost and risk. On contrary, mouse has highfecundity, short generation, and takes fewer time and money. So its nessesary to use mouse asa small animal model to do our research at first, then transfer to pig, in the end obtain skinspecifically expression human CTLA-4Ig transgenic mouse and pig, form a model systemcombined with small animal and big animal, which could contribute to futher study ofhCTLA-4Ig function and mechanism in xenotransplantation.2. ObjectiveA.To establish skin specifically expression human CTLA-4Ig transgenic mouse and pigmodel, form a model system combined with small animal and big animal, which couldcontribute to futher study of hCTLA-4Ig function and mechanism in xenotransplantation.B. To establish a population of skin specifically expression human CTLA-4Ig transgenicbama miniature pig, as a candidate alternative donor skin supply.3. MethodA. The foundation of skin specifically expression hCTLA-4Ig transgenic mousepopulation and its function research.Preparation of skin specifically expression hCTLA-4Ig transgenic mice by lentivirusvector, we will establish a population; filtration and identification of the transgenic mice byPCR; investigating the expression and distribution of transgenic protein by RT-PCR, real timePCR, ELISA, WB and immunohistochemistry; confirming the function of transgenic proteinin vitro by one-way MLC; testifying the function of transgenic protein in vivo by mouse-ratskin xenotransplantation.B. The foundation of skin specifically expression hCTLA-4Ig transgenic bama miniaturepig.Preparation of skin specifically expression hCTLA-4Ig transgenic pig by somatic cellnuclear transfer(SCNT); filtration and identification of the transgenic pigs by PCR; primarilyinvestigating the expression and distribution of transgenic protein by WB. C. The foundation of skin specifically expression hCTLA-4Ig transgenic bama miniaturepig population and its function in xenotransplantation.Generating a population of skin specifically expression hCTLA-4Ig transgenic bamaminiature pig; filtration and identification of the transgenic pigs by PCR; investigating theexpression and distribution of transgenic protein by ELISA and WB; confirming the functionof transgenic protein in vitro by one-way MLC; testifying the function of transgenic protein invivo by pig-rat skin xenotransplantation; examining the histological change of skin graft afterxenotransplantation by paraffin section HE staining.4. ResultsA. Skin specifically expression hCTLA-4Ig transgenic mice population have beenobtained, and the distribution and bioactivation of transgenic protein has been confirmed.PCR results show that8founder mice were obtained, and transgenic founder mice perinjected and transferred eggs was6.3%; cross breeding the8founder mice and sift by PCR,we obtained a skin specifically expression hCTLA-4Ig transgenic mice population; RT-PCRand WB shows the transgenic hCTLA-4Ig exhibited strictly skin-specific expression; IHshows that, too, and the transgenic hCTLA-4Ig do not interfere the development and patternof skin tissue; ELISA shows that hCTLA-4Ig is excreted, and it can be detected in serum,andits concentration comes to11.87μg/ml; one-way MLC confirms the transgenic protein caninhibit the proliferation of lymphocytes in vitro; mouse-rat skin xenotransplantation testify thebioactivity of transgenic protein in vivo, which prolongs the survival of skin grafts.B. Skin specifically expression hCTLA-4Ig transgenic bama miniature pig populationhave been estabilished, and the distribution and bioactivation of transgenic protein has beenconfirmed.Skin specifically expression hCTLA-4Ig transgenic bama miniature pig have beenobtained by SCNT, and transgenic founder pigs per injected and transferred eggs was0.75%;making the founder pigs copulate with wild-type pigs, and cross breeding the F1generation,sift by PCR, we obtained a skin specifically expression hCTLA-4Ig transgenic bamaminiature pig population;32pigs are positively transgened in59of F1generation by PCR;ELISA shows that hCTLA-4Ig is excreted, so that it can be detected in serum, and theconcentration of5randon F1generations range from5.61μg/ml to9.56μg/ml; WB showsthat transgenic protein hCTLA-4Ig is strictly keratin-targeted tissue(skin, cornea) specific expression; one-way MLC confirms the transgenic protein can inhibit the proliferation oflymphocytes in vitro; pig-rat skin xenotransplantation testify the bioactivity of transgenicprotein in vivo, which prolongs the survival of skin grafts; paraffin section HE staining ofskin grafts10days after xenotransplantation shows that hCTLA-4Ig inhibits the proliferationof lymphocytes and decrease the inflammation in grafts.5. ConclutionsA. Skin specifically expression hCTLA-4Ig transgenic mouse and bama miniature pigpopulation are generated, forming a model system combined with small animal and biganimal, which could contribute to futher study of hCTLA-4Ig function and mechanism inxenotransplantation.B. The bioactivation of transgenic hCTLA-4Ig protein has been confirmed in vivo and invitro, which indicate that skin specifically expression hCTLA-4Ig transgenic mouse and bamaminiature pig population not only can provide animal model for further study of hCTLA-4Igfunction and mechanism,but also can provide donor skin for clinic and animal model forimmune tolerance research in the future.更多还原