【作者】 姚旭东；

【导师】 马腾骧； 王广有；

【作者基本信息】 天津医科大学 ， 泌尿外科学， 2002， 博士

【摘要】 猪对人的异种器官移植被认为是解决同种移植长期器官短缺的一种方法。人体内存在针对远缘动物细胞表面多糖分子的天然抗体，此类抗体结合抗原在移植物血管内皮激活补体系统和凝血系统引发超急性排斥反应(HAR)，是异种器官移植成功的最大障碍。 一些表达人补体调节蛋白如CD55(DAF)和CD59的转基因猪已被建立，这些转基因猪可以(至少是部分地)耐受人天然抗体结合物激活补体后的袭击。然而，也许是内皮细胞固有的异质性，在整个成年供体动物血管树中，特异性高表达的转(人)基因很难达到；另外，在许多实践研究中，转基因的普遍表达将有可能与(宿主)生理不协调一致，甚或对宿主有害，生物学家由此要限制转基因在特异细胞，如内皮细胞(EC)中表达。因此，在内皮细胞中选择特异性高表达的启动子，也就成为异种移植转基因研究的重要内容之一。人细胞间粘附分子2(ICAM-2)启动子是近年来研究较多的一内皮细胞的特异启动子，能够在体内、外驱动外源基因在血管内皮细胞高表达。另外，更多的研究还表明，联合转基因动物较单基因动物显示了更强的抗超急排斥反应能力。 基于目前研究观点，我们课题组将建立在血管内皮细胞特异表达人双补体调节蛋自(CD59和DAF)的转基因猪，构建携有人ICAM-2启动子的CD59、DAF基因的特异表达载体，以及在猪血管内皮细胞(PEC)检测重组基因的表达，是建立此转基因猪的上游工作。我们课题组业已完成的工作：①从人血基因组中克隆了ICAM-2启动子片段，克隆了CD59基因第一内含子起增强子作用的DNA片段，与国外惠赠的CD59cDNA基因重组，构建了pcDNA3-En-ICAM2-CD59cDNA表达载体及不含增强子pcDNA3ICAM2-CD59cDNA表达载体。②类似上述的方法，构建了人ICAM-2作启动子的DAF基因表达载体pcDNA3-ICAM2-Intronl-DAFcDNA。③分别构建了检测ICAM-2启动子活性及包括内含子片段在内的启动子活性的绿色荧光蛋白报告基因。④培养并鉴定猪血管内皮细胞，培养猪耳成纤维细胞。用脂 天津医科大学博士学位论文 质体包裹质粒转染方法，将上述表达载体及报告基因分别转染猪血管内皮细 胞及成纤维细胞。⑤通过荧光显微镜检测报告基因的表达：通过流式细胞仪， 对CD59表达载体在PEC中瞬时表达进行了检测；通过流式细胞仪和RT－PCR 方法 对抗G4 18转pcDNA3工 一ntronl－DAFcDNA表达载体的PEC进行 表达测定。⑤筛选、克隆表达DAF蛋白的PECS，利用与含正常人血清M199 共孵育方法，检测乳酸脱氢酶漏出率，评价表达DAF蛋白PECs抗人血清溶 破的功能情况。作者在此课题中，侧重完成以下工作及研究。 1．CD59基因特异表达载体的构建 方法恨据文献ICA＼I－2启动于、CD59基因增强子序列，设计两对引物序列； 利用PCR方法从人血基因组扩增得到ICAM－二启动于、CD59基因的一段增 强子片段，经电泳分离、切胶回收纯化得到上述片段，分别双酶（Bamhl／KPul）、 单酶 Hindlll）切这两条片段，纯化回收备用；双酶切（Bgl 11、kpn I） pCDNA3（D59真核表达载体，经电泳分离、切胶口收纯化得到不含病毒启 动子、含筛选基因Neo的一段pcDNA3－CD59cDNA作为载体序列；ICAM上 启动于片段先与载体进厅连接反应后转化细菌，阳性转化菌质粒抽提及酶切 鉴定．得到pCDNA3－ICAMZ－CD59质粒：再单酶（Hifldth）切此质粒，纯化该 载体与CD59增强子片段连接，转化细菌、抽提质粒。 结果得到pcDNA3工n1C人＊－CD59表达载体．以EcoRI消化此重组质粒， 产上5、42kb及05kb丙片段，以Hindtti消化重组质粒，得到5．47kb及0．45kb 两片段，以Hind山／Kpnl双酶切质粒时，出现0．4kb、0．45kb． 5o7kb三条片 段，上还结果完全符合设计要求：对插入子分别进厅测序，结果符合基因库 序列。 结论表明含人ICANIZ 启动子的CD59表达载体pCDNA3二－CD59、 pcDNA3－En－ICAMZ－CD59构建获得成功。 2．DAF基因特异表达载体的重建 方法 双酶切本室己将ICAM－2 启动子与DAFCDNA 重组的原核质粒 pGEMJZtLDAF，得到含人ICANI上启动子及DAFCDNA等序列的插入片段 天津医科大学博士学位论文 门．7kb＼ 双酶切 pCDNA3真核表达载体，得到不含病毒启动子、含筛选基 因Neo的一段DNA作为载体序列（4．4kb）；两段DNA进行连接反应后转化 细菌；阳性转化菌落质粒抽提及酶切鉴定。根据人的ICAM－2 启动子、 DAFCDNA序列，设计引物行PCR特异扩增检验。 结果特异性三组酶切重组表达载体，产生符合设计的相应条带：KR扩增 出特异的 3 3 obp及 1．7kb的 DNA片段，与设计要求一致。 结论 含 人ICAM－2 启 动子 ?更多还原

【Abstract】 The chronic shortage of human organs, tissues and cells for transplantation has inspired research on the possibility of using animal donor tissue instead. Transplantation over a species barrier is associated with rejections which are difficult to control. Vascular endothelium is the most immediate barrier between the xenogeneic donor organ and host immune and nonimmune defense systems. Thus, these cells are the prime targets for such genetic modifications.Transgenic pigs have been made that express human complement regulatory proteins such as CDS9 and DAF, which allow these pigs to withstand, at least partly, the attack of human complement following natural antibody binding and complement activation. However, specific high-level expression of transgenes throughout the vascular tree in adult animals has proved difficult to achieve, perhaps because of the inherent heterogeneity of endothelium. In all these cases promoters with activity in a wide range of tissues were used. In cases in which a general expression of the thansgene may be incompatible with normal physiology, or even lethal to the host, one would want to restrict the expression of the transgene to the cell type of interest, e.g. endothelial cells(ECs). Hence, selecting the strong endothelial-specific promoters become one of most important parts in the research of xenotransplantation. The promoter of intercellular adhesion molecule-2 (ICAM-2) has been a endothelial cell-specific promoter studied currently and could drive transgen to expresse high-level in vivo and in vitro. Moreover, many studies suggest that engineering donor pigs to express multiple molecules that address different humoral components of xenograft rejection represents an important step toward enhancing xenograftsurvival and improving the prospect of clinical xenotransplantation.According to the above, we want to establish the transgenic pigs expressing two human complement regulator}’ proteins that can prevent rejection .The upstream job of the pigs is to construct the effective specific vectors expressing CD59 andDAF gene. In present study, we cloned the promoter sequence of ICAM-2 fromthe human blood genome, and the fragments of intronl of CD59 and DAF genesrespectively. ICAM-2 promoter acted as a promoter, and the fragment of intron 1acting an enhancer, the expression vectors of CD59 and DAF gene wereconstructed respectively. Using the method of liposomes transfection, the vectorswere transferred into pig aorta endothelial cells and the expression were measuredby flow cytometer and PT-PCR. Finaly, the endothelial cells expressing resistantDAF proteins were incubated with human serum, and the function of thetransfeutants resisting lysis from human serum were measured.l.The construction of the expression vectors of CD59 gene using ICAM-2promoterMethods Produced ICAM-2 promotor fragment and CD59-intronl fragment byPCR from the human blood genome, then inserted these fragments into apcDNA3-CD59 eukaryotic expression vector. Digested this recombinant plasmidwith the special restriction endonucleases (for example.EcoR I /HindLU) . TheICAM-2 promoter and CD59-intronl fragments was identified by PCR, and wassequencd.Results Productions by digestion accord with the design. The two DNAfragment sequences. ICAM-2 promoter and CD59-intronl fragments, are thesame as the frames of the gene bank.Conclusions The specific expression vector of CD59 gene was constructedsuccessfully.2. The reconstruction of the specific expression vectors of DAF geneMethods Cut the pGEM-7Zf -DAF plasmid with restriction endonucleases,andobtaining the recombinant human DAF gene which containing ICAM-2 promotorfragment, DAFcDNA and polyA. In the same way, obtained the pcDNASfragment expression vector. Above two DNA fragments performed recombinantreaction and the production was transformed into reception germs. Theplasmids pick-uped from positive transformed germs were identified by the更多还原