绿色荧光蛋白在α-1,3半乳糖基转移酶敲除猪组织器官的表达分析

猪是人类异种器官移植的理想供体,然而猪-人的异种器官移植会产生剧烈的排斥反应。虽然已制备的α-1,3半乳糖基转移酶基因敲除（Galactosyltransferase gene knockout, GTKO）猪可有效缓解猪-人异种器官移植引起的超急性免疫排斥,但缺少报告基因直观示踪移植后的细胞迁移及器官排斥状态。本文将CAG启动子驱动增强型绿色荧光蛋白（Enhanced green fluorescent protein, EGFP）的表达载体导入GTKO猪耳成纤维细胞,通过体细胞核移植技术制备了EGFP猪。利用双荧光蛋白观测镜、荧光显微镜及定量PCR扩增观察、检测和分析克隆猪各组织器官中EGFP蛋白和转录本的表达状况。结果显示,EGFP蛋白及转录本在克隆猪各组织器官中均有表达,但在肝脏和中枢神经系统中表达较弱。本文成功获得了各组织器官表达EGFP的GTKO猪,为EGFP示踪异种细胞组织移植奠定了基础。     

基因修饰猪作为异种器官移植供体的研究进展

器官移植是治疗脏器器官终末病变的根本方法,目前器官移植的最大障碍是供体器官的缺乏。异种器官移植为解决上述难题打开了一扇门。猪与人类在解剖学和生理学等方面非常相似,是理想的器官移植的供体。基因修饰猪在异种器官移植中有着重要的应用,该文就猪–人异种器官移植发生免疫排斥的机制及其对策、器官移植后的生理功能以及潜在的病毒感染风险等方面的研究进行了综述和讨论,为异种器官移植提供理论基础。     

中国版纳小型猪近交系动脉异种移植靶抗原研究

目的　研究中国版纳小型猪近交系动脉的异种靶抗原α -Gal的分布和半定量分析 ,为研究异种移植超急性排斥反应和异种生物材料提供资料。方法　通过亲和免疫组织化学法和图象分析对 10头版纳小型猪的四级动脉进行α Gal的分布和半定量研究。结果　 (1)各级血管组织中血管内皮细胞阳性表达明显 ,弹性纤维、胶原纤维和平滑肌细胞未见表达 ,外膜有微弱表达 ;(2 )图象分析显示 ,管径越细 ,其内皮细胞的表达越高 (P <0 .0 1) ,各级动脉内皮细胞阳性表达的面积密度比的组间差异显著 (P <0 .0 1) ,t检验发现每两组间的动脉内皮细胞阳性表达面积密度比相差显著 (P <0 .0 1)。结论　 (1)版纳小型猪动脉内皮细胞均有异种抗原α Gal分布 ,但是分布不均匀 ;(2 )微血管血栓是异种移植超急性排斥反应中的重要环节 ,小型猪的异种器官移植、彻底解决异种移植超急性排斥反应的关键是防治微血管栓塞     

转人源免疫球蛋白猪胚胎的研究进展

转基因猪能够为人类提供可移植的异种器官,从而缓解临床医学应用上供体移植器官短缺的压力.本文综述了异种器官移植后发生免疫排斥的研究进展,讨论了应用原核显微注射法和精子栽体法生产转人源免疫球蛋白基因(DAF、MCP、CD59)巴马香猪胚胎的方法,力争为解决异种移植的技术"瓶颈"提供技术支撑,为后续的异种器官移植做初步的研究.     

转基因猪异种器官移植应用前景

转基因猪能为人类提供可移植的异种器官,解决移植器官短缺的问题.但是,异种移植后产生的免疫排斥反应,主要包括超急性排斥反应和迟发性排斥反应,它们最终将导致移植物的致命损伤,是限制异种器官广泛应用的最大障碍.最近的一些研究,在克服免疫排斥方面已经取得了可喜的成果.作为异种器官供体,猪携带的内源性逆转录病毒也可能对人体产生潜在的威胁.迄今,还没有研究证明其不会感染人类机体.本文针对异种器官移植后的免疫排斥发生的机理,克服免疫排斥的方法以及逆转录病毒的潜在感染等方面进行了综述和讨论.     

异种移植的病毒安全性研究进展

猪-人异种移植有望解决人源器官短缺的严重问题。然而,以前病毒（provirus）形式整合入猪基因组中的猪内源性反转录病毒（porcine endogenous retrovirus,PERV）难以去除,PERV有可能通过异种移植传播给人类,甚至产生新的病毒性疾病。本文回顾了PERV与异种移植病毒安全性及我国特有小型猪中PERV的相关研究。     

遗传修饰猪模型在生物医学及农业领域研究进展及应用

猪在解剖结构、代谢、生理生化等特征方面比啮齿类动物更接近人类,因此在模拟某些人类疾病以及提供异种移植器官等方面具有其他动物不可替代的优势,是理想的人类疾病动物模型和异种器官的供体。另外,猪作为我国畜牧业最重要的物种之一,猪的品种改良、疫病防控以及动物福利等问题都与人民生活息息相关。本文主要介绍了遗传修饰猪模型在分子育种、人类疾病模型以及异种器官移植领域的研究进展及未来应用前景,希望增进相关领域研究人员对基因编辑等前沿技术的了解,理解遗传修饰猪模型在生命科学研究中的重要意义。     

绿色荧光蛋白在α-1,3半乳糖基转移酶敲除猪组织器官的表达分析

猪是人类异种器官移植的理想供体,然而猪-人的异种器官移植会产生剧烈的排斥反应。虽然已制备的α-1,3半乳糖基转移酶基因敲除(Galactosyltransferase gene knockout,GTKO)猪可有效缓解猪-人异种器官移植引起的超急性免疫排斥,但缺少报告基因直观示踪移植后的细胞迁移及器官排斥状态。本文将CAG启动子驱动增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)的表达载体导入GTKO猪耳成纤维细胞,通过体细胞核移植技术制备了EGFP猪。利用双荧光蛋白观测镜、荧光显微镜及定量PCR扩增观察、检测和分析克隆猪各组织器官中EGFP蛋白和转录本的表达状况。结果显示,EGFP蛋白及转录本在克隆猪各组织器官中均有表达,但在肝脏和中枢神经系统中表达较弱。本文成功获得了各组织器官表达EGFP的GTKO猪,为EGFP示踪异种细胞组织移植奠定了基础。     

中国内江猪肾脏的的解剖学研究

根据医学异种移植研究的需要,选取５－６月龋中国内江猪１９头,处死后甲醛固定肾脏标本,进行测量并与国人肾脏解剖学资料对比,以了解中国内江猪肾脏解剖学特点及与人肾脏的差别。结果表明内江猪肾脏与国人肾脏无大的解剖学差异。并具有分支型、速走型肾动脉出现率低的优点。认为从解剖学角度看,中国内江能脏完全符合异种移植的要求。     

XENOTRANSPLANTATION, CONSENT AND INTERNATIONAL JUSTICE

The risk posed to the community by possible xenozoonosis after xenotransplantation suggests that some form of 'community consent' is required before whole organ animal-to-human xenotransplantation should take place. I argue that this requirement places greater obstacles in the path of ethical xenotransplantation than has previously been recognised. The relevant community is global and there are no existing institutions with democratic credentials sufficient to establish this consent. The distribution of the risks and benefits from xenotransplantation also means that consent is unlikely to be forthcoming. Proceeding on the basis of hypothetical consent to a package of global health measures that includes xenotransplantation, as Rothblatt has recently advocated, is more problematic than she acknowledges. Given that it may place the lives of citizens of poor nations at risk to benefit the citizens of wealthy nations, xenotransplantation raises significant questions of international justice.     

In pursuit of xenoreactive antibodies: where has it gotten us?

Numerous studies have aimed to overcome the barrier to xenotransplantation posed by xenoreactive antibodies and the antigens they recognize. Whether this work will eventually lead to the widespread clinical application of xenotransplantation remains unknown. However, the benefits of this research are already substantial, with research leading to dramatic new developments in fields other than xenotransplantation. Our understanding of natural immunity, particularly the nature and function of natural antibodies, has taken quantum leaps forward, with far-reaching implications. Our improved understanding of the immune response to xenografts has proven invaluable in the characterization of the human immune reaction to commonly used biological therapeutics of xenogeneic origin. Our understanding of cell surface carbohydrates and our ability to modify these carbohydrates in living animals has advanced substantially, with implications for diseases such as cancer and autoimmunity. With this in mind, it is argued that continued work in xenotransplantation is of great value, not only because of the great potential benefits of xenotransplantation, but also because of the more certain benefits that arise from setting our sights on a difficult challenge.     

Xenotransplantation: regulatory challenges

During 1999-2000, the US government published three xenotransplantation policy/guidance documents, one by the Public Health Service and two by the Food and Drug Administration (FDA). The FDA also held two public meetings of the xenotransplantation subcommittee of the Biological Response Modifiers Advisory Committee to discuss particular issues in xenotransplantation.     

Blood brothers: carbohydrates in xenotransplantation and cancer immunotherapy

Carbohydrate antigens have a central role in the hyperacute rejection of animal-to-human organ grafts (xenotransplantation) and they are emerging in importance in the immunotherapy of cancer. This article traces the historical origins of the discovery of key carbohydrate antigens and explores the future impact of recent technological advances of the field of glycobiology as it relates to xenotransplantation and cancer.     

Infection of nonhuman primate cells by pig endogenous retrovirus

The ongoing shortage of human donor organs for transplantation has catalyzed new interest in the application of pig organs (xenotransplantation). One of the biggest concerns about the transplantation of porcine grafts into humans is the transmission of pig endogenous retroviruses (PERV) to the recipients or even to other members of the community. Although nonhuman primate models are excellently suited to mimic clinical xenotransplantation settings, their value for risk assessment of PERV transmission at xenotransplantation is questionable since all of the primate cell lines tested so far have been found to be nonpermissive for PERV infection. Here we demonstrate that human, gorilla, and Papio hamadryas primary skin fibroblasts and also baboon B-cell lines are permissive for PERV infection. This suggests that a reevaluation of the suitability of the baboon model for risk assessment in xenotransplantation is critical at this point.     

Intracellularly expressed single-domain antibody against p15 matrix protein prevents the production of porcine retroviruses

The presence of porcine endogenous retroviruses presents a potential risk of transmission of infectious diseases (xenozoonosis) if tissues and organs from genetically modified pigs are to be used in xenotransplantation. Here, we report that intracellular expression of a llama single-domain antibody against p15, the matrix domain protein of the porcine endogenous retrovirus Gag polyprotein, blocks retrovirus production, providing the possibility of eliminating the risk of infection in xenotransplantation.     

Xenotransplantation and other means of organ replacement

Exciting new technologies, such as cellular transplantation, organogenesis and xenotransplantation, are thought to be promising approaches for the treatment of human disease. The feasibility of applying these technologies, however, might be limited by biological and immunological hurdles. Here, we consider whether, and how, xenotransplantation and various other technologies might be applied in future efforts to replace or supplement the function of human organs and tissues.     

The resurgent landscape of xenotransplantation of pig organs in nonhuman primates

Organ shortage is a major bottleneck in allotransplantation and causes many wait-listed patients to die or become too sick for transplantation. Genetically engineered pigs have been discussed as a potential alternative to allogeneic donor organs. Although xenotransplantation of pig-derived organs in nonhuman primates(NHPs) has shown sequential advances in recent years, there are still underlying problems that need to be completely addressed before clinical applications, including(i) acute humoral xenograft rejection;(ii) acute cellular rejection;(iii) dysregulation of coagulation and inflammation;(iv) physiological incompatibility; and(v) cross-species infection. Moreover, various genetic modifications to the pig donor need to be fully characterized, with the aim of identifying the ideal transgene combination for upcoming clinical trials. In addition, suitable pretransplant screening methods need to be confirmed for optimal donor-recipient matching, ensuring a good outcome from xenotransplantation. Herein, we summarize the understanding of organ xenotransplantation in pigs-to-NHPs and highlight the current status and recent progress in extending the survival time of pig xenografts and recipients. We also discuss practical strategies for overcoming the obstacles to xenotransplantation mentioned above to further advance transplantation of pig organs in the clinic.     

Production of genetically modified pigs expressing human insulin and C-peptide as a source of islets for xenotransplantation

Transgenic Research - Islet xenotransplantation is a promising treatment for type I diabetes. Numerous studies of islet xenotransplantation have used pig-to-nonhuman primate transplantation models....