异种肾移植：生理学研究的现状及发展趋势

器官移植是治疗各类终末期器官疾病最有效的手段,为了解决器官移植中供者短缺的问题,人们开始探究异种移植。目前人们普遍关注异种移植排斥反应及病毒感染相关的问题,在异种肾移植生理学方面的研究较少。肾脏通过产生促红细胞生成素（EPO）、肾素,激活维生素D来执行内分泌功能。虽然这些途径在同种移植中通常保存良好,但物种特有的差异,特别是猪和非人灵长类动物之间的差异,仍然可能会影响移植器官的生理机能。本文尝试从猪与人的EPO、肾素-血管紧张素-醛固酮系统（RAAS）、有活性的维生素D₃等在异种移植后的作用变化等方面进行阐述,旨在为异种移植亚临床研究提供参考。     

Engineering pigs for xenotransplantation

Xenotransplantation using tissues and organs from genetically modified pigs has the potential to fulfil the serious shortage of material available for human transplantation. However, considerable immunological barriers must be overcome before such discordant grafts can be used. The generation of α(1,3)‐galactosyltransferase knock out pig lines has surmounted the initial obstacle of hyperacute rejection. Further improvements are now necessary to provide adequate protection against acute humoral xenograft rejection. The expression of human complement regulator transgenes in existing transgenic animals is inadequate, and a means of achieving abundant uniform expression in the organ is required. Recent findings have also highlighted the need for additional transgenes to alleviate incompatibilities between the porcine and human anticoagulation and anti‐inflammatory systems. Generating multi‐transgenic animals by breeding individual transgenic animals is inefficient and incurs several problems, including: independent Mendelian segregation of multiple transgenes; failure of different transgenes to co‐express at the right time or in the correct tissue; and an increased risk of insertional mutagenesis due to multiple integration events. A novel approach is therefore required to produce what are likely to be increasingly complex multi‐transgenic animals. Ideally, all transgenes should reside at a single Mendelian locus. They should not be subject to the position effect. There should be no limitation on transgene size to allow inclusion of substantial regulatory regions, or genes as multiple copies to obtain abundant expression. Finally, insertional mutagenesis should be excluded if possible. One possible way to achieve these is to use artificial chromosome vectors. De novo formed human artificial chromosomes are being developed as vectors for human gene therapy. The feasibility of this and alternative approaches to deliver sets of xenoprotective transgenes into pigs is being explored. This work is being carried out in close collaboration with the Xeno‐Forschergruppe (FOR 535) and is supported through funding provided by the DFG.     

The case for xenotransplantation

The availability of organs and cells from deceased humans for transplantation is not meeting the demand. Xenotransplantation, specifically the transplantation of organs and cells from genetically engineered pigs, could resolve this problem. Diabetic monkeys have remained normoglycemic and insulin‐independent after pig islet transplantation for >one yr, and a pig heterotopic (non‐life‐supporting) heart transplant recently reached the one‐yr milestone in a baboon. With these encouraging results, why is it that, with some notable exceptions, research into xenotransplantation has received relatively little support by industry, government funding agencies, and medical charitable foundations? Industry appears reluctant to support research that will take more than two to three yr to come to clinical trial, and the funding agencies appear to have been “distracted” by the current appeal of stem cell technology and regenerative medicine. It has only been the willingness of living donors to provide organs that has significantly increased the number of transplants being performed worldwide. These altruistic donations are not without risk of morbidity and even mortality to the donor. Although with the best of intentions, we are therefore traversing the Hippocratic Oath of doctors to “do no harm.” This should be a stimulus to fund exploration of alternative approaches, including xenotransplantation.     

Genetic engineering for xenotransplantation

Xenotransplantation is being pursued vigorously to solve the shortage of allogeneic donor organs. Experimental studies of the major xenoantigen (Gal) and of complement regulation enable model xenografts to survive hyperacute rejection. When the Gal antigen is removed or reduced and complement activation is controlled, the major barriers to xenograft survival include unregulated coagulation within the graft and cellular reactions involving macrophages, neutrophils, natural killer (NK) cells, and T lymphocytes. Unlike allografts, where specific immune responses are the sole barrier to graft survival, molecular differences between xenograft and recipient that affect normal receptor-ligand interactions (largely active at the cell surface and which may not be immunogenic), are also involved in xenograft failure. Transgenic strategies provide the best options to control antigen expression, complement activation, and coagulation. Although the Gal antigen can be eliminated by gene knockout in mice, that outcome has only become a possibility in pigs due to the recent cloning of pigs after nuclear transfer. Instead, the use of transgenic glycosyl transferase enzymes and glycosidases, which generate alternative terminal carbohydrates on glycolipids and glycoproteins, has reduced antigen in experimental models. As a result, novel strategies are being tested to seek the most effective solution. Transgenic pigs expressing human complement-regulating proteins (DAF/CD55, MCP/CD46, or CD59) have revealed that disordered regulation of the coagulation system requires attention. There will undoubtedly be other molecular incompatibilities that need addressing. Xenotransplantation, however, offers hope as a therapeutic solution and provides much information about homeostatic mechanisms.     

The pig-to-primate immune response: relevance for xenotransplantation

Abstract:  Background:  The allotransplantation of some solid organs can be associated with a graft‐vs.‐host (GVH) response from the activity of donor B or T cells. We have investigated whether there is a risk of a GVH response following pig‐to‐primate organ xenotransplantation. Methods:  The responses of 16 pigs (six farm‐housed wild‐type and five wild‐type housed under high herd health conditions [all designated WT], and 5 α1,3‐galactosyltransferase gene‐knockout [GT‐KO] housed under high herd health conditions) to human (n = 6) and baboon (n = 6) peripheral blood mononuclear cells (PBMC) were determined. Assays included flow cytometry, complement‐dependent cytotoxicity, and mixed lymphocyte reaction. Results:  Anti‐primate cytotoxic IgM antibodies were detected in the sera of all pigs, but anti‐primate IgG antibodies were minimal. All pigs demonstrated a cellular proliferative response to primate PBMC that was equivalent to, or greater than, the allo response. The strength of the pig‐to‐primate GVH responses was proportional to the health status of the pigs, those from a high health status herd, particularly from a specific pathogen‐free herd maintained under clean husbandry conditions, where colonization of the gastrointestinal tract may be reduced, having lower responses. Conclusions:  After pig organ transplantation in a primate, if the organ is from an early‐weaned, early‐segregated GT‐KO pig, the strength of a GVH response is likely to be relatively weak. Although not investigated here, any GVH response is likely to be suppressed by the immunosuppressive therapy administered to the recipient to suppress the anti‐donor immune response.     

Solid organ xenotransplantation at the interface between research and clinical development: Regulatory aspects

During the last years, progress has been made in survival and function of pig-to-non-human primate organ xenotransplantation using organs from genetically modified pigs and immunosuppression regimens that are clinically acceptable. This, together with increased insights into a low risk of pig-to-human transmission of porcine endogenous retrovirus, has opened the perspective of starting with first-in-human trials with xenogeneic organs. The regulatory path to clinical development is complex. Unlike an organ from human donors, an organ from pigs, either genetically modified or wild-type pigs, is considered a medicinal product for human use and hence is under regulatory oversight, in the United States by the Food and Drug Administration and in Europe by the national competent authorities of the member states as well as the European Medicines Agency. Related to the status of medicinal product, “(current) good practices” apply in the process of generating a xenogeneic organ through to the transplantation into a patient and life-long follow-up. In addition, guidances for xenotransplantation products and genetically modified organisms do apply as well. This commentary focuses on regulatory aspects of transplantation of organs from genetically modified pigs into humans, with the intention to facilitate the interactions between regulatory agencies and institutions (sponsors) in research and clinical development of these organs, to support the perspective of speeding up the process with a proper entry in clinical application, to fill an unmet medical need in patients with end-stage organ disease.     

袖套法异种心脏移植模型

采用Heron袖套法进行异种（小鼠→大鼠）心脏移植，异位移植于颈部皮下，供体主动脉接受体颈内动脉，供体肺动脉接受体项外静脉，并对手术方法进行了部分改进。进行正式手术22次，成功率86％，移植后平均存活时间2．10±0．80天；该方法简单、迅速、可靠，移植于颈部易于观察，对于超急性排斥反应的观察以及免疫耐受的诱导，抗免疫排斥药物筛选是一位得推广的动物模型。     

Viral gene transfer for xenotransplantation

In order to overcome the major immunological barriers to xenotransplantation, genetic strategies have to be developed that ensure long‐term engraftment of the organ. To this end, immune‐modulatory transgenes have to be efficiently expressed and/or the expression of xeno‐relevant porcine genes (xeno‐epitopes) has to be silenced. Viral vectors are powerful tools for modulating expression of foreign genes in organs and even the whole animal. Using lentiviral gene transfer in early embryos, gain‐of‐function models can be generated with high efficacies in pig (lentiviral transgenesis). Through combination of lentiviral transgenesis and RNA interference (RNAi) loss‐of‐function models for xenotransplantation can be generated. Our goal is to combine lentivector‐mediated RNAi directed against α‐galactosyltransferase (α‐GT) with immune‐modulatory approaches to finally generate multitransgenic pigs. Furthermore, sensitivity to human serum inactivation of porcine endogenous retrovirus produced by pig cells with reduced levels of α‐GT expression was analysed.     

The prospects for renal xenotransplantation

Summary: Xenotransplantation of non-human organs into human recipients has long been proposed as a possible strategy to overcome the acute shortage of donor organs. However, vascular organ transplants to humans from phylogenetically disparate species such as the pig are not currently possible due to a rapid rejection process termed hyperacute rejection. This process is initiated by the binding of host pre-formed 'natural antibodies' to the donor vascular endothelium, activation of the host complement system and activation or injury of the donor endothelial cells, leading to intravascular coagulation and loss of the graft due to ischaemic necrosis within minutes to hours of engraftment. Prevention of natural antibody binding and complement activation is viewed as paramount to preventing hyperacute rejection. Even if hyperacute rejection can be prevented, further barriers to successful discordant xenografts such as delayed xenograft rejection and a donor-directed cell-mediated rejection process will still represent major obstacles. This review examines recent advances being made in the various areas of xenograft research and the potential clinical application of pig-to-human xenografts that these strategies may bring.     

Cloning and transgenesis in mammals: Implications for xenotransplantation

Availability of suitable organs for transplantation remains of major concern and projections indicate that the problem will continue to increase. Therefore, alternatives to the use of human organs for transplantation, continue to be explored including use of stem cells, artificial organs, and organs from other species (xenotransplantation). In xenotransplantation, the species of choice remains the pig due to its physiological similarities to humans, reduced costs, ease of manipulation, and reduced ethical concerns to its use. However, in order to develop pig organs that are suitable for xenotransplantation, complex genetic modification need to be undertaken. These modifications require the introduction of precise genetic changes into the pig that can only be accomplished at this time using somatic cell nuclear transfer. We cover in this review advances in transgenic manipulation and cloning in swine and how the development of these two technologies is critical to the eventual utilization of the pig as a human organ donor.     

Heterotopic cardiac xenotransplantation: fish-to-rat

The purpose of this study was to determine whether a vascularized organ transplanted from a teleost fish to a rodent would be hyperacutely rejected. Previous reports describing the results of discordant xenotransplantation are almost exclusively across orders within the class Mammalia. We chose a species combination that crosses many phylogenetic barriers (i.e. species, genus, family, order, and class) as well as several hundred million years on an evolutionary timescale. Because no published methodology existed, we developed a microvascular surgical method for fish (tilapia)-to-rat heterotopic cardiac xenotransplantation. To minimize the blood pressure to which the graft would be exposed, the tilapia heart was placed on the venous side of the rat circulation between the left kidney and the inferior vena cava by end-to-side anastomoses of the donor aorta to the recipient inferior vena cava and by end-to-end anastomosis of the donor sinus venosus to the recipient left renal vein. Tilapia hearts were rejected hyperacutely, based on both routine histopathological examination and immunofluorescent staining for immunoglobulin and complement, but rejection required hours rather than minutes.