异种移植超急性排斥反应的机制

早在１９０５年，法国医生Ｐｒｉｎｃｅｔｅａｕ就进行了世界上第一例临床异种移植手术。近几年来，由于人同种移植飞速发展，导致供者器官再次严重短缺，所以，自９０年代初，异种移植再次引起人们的兴趣。美国匹兹堡Ｓｔａｒｚｌ等［１］预测，器官移植的最终发展趋势将逐渐走向使用动物器官，通过基因工程和其它调节供者器官的方法，将使异种移植的广泛开展成为可能。一、超急性排斥反应目前，临床异种移植最常用、最理想的供者动物是猪。猪与人类的非协调性异种移植的最大障碍是由体液免疫造成的、发生于移植后数分钟至数小时的超急性排…     

异种移植中转基因动物策略研究进展

近年来 ,在同种移植研究的基础上 ,异种移植的研究也进行了大量的工作 ,突出的成绩表现在异种移植免疫障碍阶段的划分和定义 ,即超急排斥反应、急性血管排斥 (有时也称延迟排斥 )、细胞排斥或慢性排斥、生理功能及其它。根据异种移植免疫学理论 ,利用转基因技术 ,建立转基因动物克服超急免疫排斥是异种移植研究的又一进步 ,显示转基因动物具有很大的发展前景 ,使异种移植走进临床成为可能。本文将就近年异种移植免疫学理论及根据其机制、利用转基因动物克服免疫排斥研究状况作一综述。     

异种移植中转基因动物策略研究进展

近年来,在同种移植研究的基础上,异种移植的研究也进行了大量的工作,突出的成绩表现在异种移植免疫障碍阶段的划分和定义,即超急排斥反应,急性血管排斥（有时也称延迟排斥）,细胞排斥或慢性排斥,生理功能及其它,根据异种移植免疫学理论,利用转基因技术,建立转基因动物克服超急免疫排斥是异种移植研究的又一进步,显示转基因动物具有很大的发展前景,使异种移植走进临床成为可能,本文将就近年异种移植免疫学理论及根据其机制,利用转基因动物克服免疫排斥研究状况作一综述。     

异种心脏移植急性血管性排斥反应期组织因子与凝血的关系

目的探讨异种心脏移植后急性血管性排斥反应期移植心心肌中供、受者的组织因子与凝血的关系。方法供者为清洁级豚鼠,受者为清洁级SD大鼠。随机配对平均分为6组。建立异种心脏移植急性血管性排斥反应期动物模型。分别于移植术后0(未行心脏移植)、4、8、12、16和24h切取移植心脏,使用免疫组织化学染色法观察移植心的心肌凝血程度;同时采用逆转录聚合酶链反应(RT-PCR)监测移植心心肌中组织因子的表达。结果移植后8h,移植心心肌凝血程度明显,并随移植时间延长逐渐加重。供者组织因子mRNA表达在移植后4h最强,随后逐渐减弱,至移植后16h消失;受者组织因子mRNA表达在移植后16h出现,随后逐渐增强。结论移植心心肌中的组织因子在异种心脏移植后急性血管性排斥反应期凝血中起重要作用,其中供者的组织因子可能与凝血的激发有关,受者的组织因子可能与凝血的加重有关。     

转染人补体调节基因对猪血管内皮细胞的保护作用

供者血管内皮细胞(EC)是猪到人异种移植时引发超急性排斥反应的靶抗原的主要分布部位。利用转基因技术让猪的内皮细胞表达人补体调节基因(hDAF)，可能会增强其血管内皮细胞的防御能力，借以克服超急性排斥反应的发生，联合转染一种以上人补体调节蛋白基因可能效果更好。为此，我们进行了如下实验。     

异种肝移植凝血功能调节障碍的免疫生物学机制

器官短缺是目前困扰移植医学发展的主要难题.美国器官资源共享网络的数据显示,至2012年4月,共有110000例患者在等待捐献器官,而其中肝病患者就有17 000例[1].我国器官短缺形势则更为严峻,每年等待移植的患者有150万人,仅能实施1万例移植[2--3],有30万人因缺乏供肝而死亡.开展异种器官移植研究,用动物器官桥接甚至代替人体器官进行移植,是解决供者器官短缺的有效途径[4]. 猪是当前公认的可能用于人类的异种移植合适供者,其具有器官体积和生理指标与人类接近、繁殖速度快、适于进行基因改造和修饰等优势[5-6].目前以α-1,3-半乳糖转移酶基因敲除(GTKO)猪为供者、非人灵长类动物为受者的异种肾移植和心脏移植的移植物最长存活时间分别为83 d[7]和179 d[8],而异种肝移植的移植物存活时间仅为14 d.与肾脏和心脏移植相比,异种肝移植后不仅存在超急性排斥反应和急性排斥反应等难题,而且受者的凝血功能调节障碍更为严重,90％的受者因血小板严重减少所致的内脏出血而死亡.因此,如何纠正凝血功能调节障碍,是当前异种肝移植研究的重点和热点问题.     

异种肾移植超急性排斥反应时血液流变学的变化

采用猪-犬肾移植建立超急性排斥反应模型，以研究此时的血液流变学变化。分别在移植前、移植肾超急性排斥发生时和排斥肾切除后10～15分钟检测外周血的血液流变学参数变化。结果异种肾移植发生超急性排斥时，血小板聚集显著增高，移植肾切除后血中的血小板数和纤维蛋白原明显下降，血小板聚集也恢复原有水平。提示血小板、纤维蛋白原参与了异种移植超急性排斥反应的发生过程，而血小板聚集可能是诊断异种移植超急性排斥反应一个有价值的指标。     

异种移植诱导免疫耐受策略的研究进展

异种移植时由于需要克服强有力的免疫排斥反应，免疫抑制剂的大量应用带来的危险性远远超过了同种器官移植，因此目前认为只有诱导人体对动物器官产生特异性的免疫耐受，异种移植才有可能走向临床。本文就近年来诱导供者特异性免疫耐受的策略研究进展进行了综述。     

肾移植术后供者特异性抗体对移植肾近期效果的影响

目的 评价肾移植术后供者特异性抗体(Ds-Ab)对移植肾近期效果的影响。方法 对2001年1月至2002年7月间进行尸肾移植的92例受者，使用酶联免疫吸附(ELISA)法，检测受者血清中HLA抗体水平，随访1年。结果 16例(17．4％)受者术后出现供者特异性抗体。抗体阳性组急性排斥发生率(56．3％)高于抗体阴性组(11．9％)，P=0．000；移植肾功能延迟恢复的发生率(12．5％)与抗体阴性组(9．2％)比较，差异无显著性，P=0．102；供者特异性抗体阳性组受者发生急性排斥后，移植肾肌酐水平高于抗体阴性组或无急性排斥组。结论 供者特异性抗体与肾移植术后急性排斥有关，可能影响近期移植肾功能。     

清除补体避免猪到猕猴异种心脏移植超急性排斥反应的实验研究

目的 观察纯化的眼镜蛇毒因子(CVF)对猪到狱猴异种心脏移植超急性排斥反应的影响。方法 以幼猪为供者，施行猪到狱猴腹腔内异位心脏移植，实验组(n＝4)使用CVF完全清除受者体内补体，对照组(n＝5)不使用CVF，两个组术后均采用环抱素A、甲泼尼龙和环磷酰胺抑制排斥反应，通过检测血清C3、C4水平及总补体活性验证CVF的效果，移植心停跳时切取移植心进行病理检查。结果 在使用CVF后，实验组血清C3降为0，总补体活性CH50值也几乎为0，末发现明显毒副反应，移植猪心存活时间平均为lld，最长达13d，病理学提示均发生了延迟性异种排斥反应；对照组3个移植心在移植后60min内发生超急性排斥反应，另2个分别存活22h及6d。结论 纯化的CVF有良好的清除补体的作用，且末见明显副作用；使用CVF可克服猪到狱猴异种心脏移植超急性排斥反应的发生。     

Advances in the development of experimental composite tissue transplantation models

The preclinical experimental models of composite tissue allograft (CTA) have rapidly developed in the past years. When microsurgical techniques were established, researchers focused on immunomodulatory protocols that overcome the immunologic barrier between the allogenic donor and recipient. To test immunologic response, functional recovery, and technical feasibility, experimental CTA has been performed in different models, including rodents, large animals, and nonhuman primates. In the experimental studies, researchers are focused on tolerance-inducing strategies based on immunosuppressive protocols allowing for widespread application in the clinic. In this review, authors analyzed the current knowledge of immunologic aspects and tolerance-inducing strategies in CTA experimental models, including single components such as skin or vascularized bone allograft versus CTA containing multiple tissues such as experimental limb and face transplants, and emphasized their relevance and applicability to the clinical scenario.     

Inflammatory cytokine and catabolic enzyme expression in a goat model of intervertebral disc degeneration

Intervertebral disc degeneration is implicated as a leading cause of low back pain. Persistent, local inflammation within the disc nucleus pulposus (NP) and annulus fibrosus (AF) is an important mediator of disc degeneration and negatively impacts the performance of therapeutic stem cells. There is a lack of validated large animal models of disc degeneration that recapitulate clinically relevant local inflammation. We recently described a goat model of disc degeneration in which increasing doses of chondroitinase ABC (ChABC) were used to reproducibly induce a spectrum of degenerative changes. The objective of this study was to extend the clinical relevance of this model by establishing whether these degenerative changes are associated with the local expression of inflammatory cytokines and catabolic enzymes. Degeneration was induced in goat lumbar discs using ChABC at different doses. After 12 weeks, degeneration severity was determined histologically and using quantitative magnetic resonance imaging (MRI). Expression levels of inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], and IL-6) and catabolic enzymes (matrix metalloproteinases-1 [MMPs-1] and 13, and a disintegrin and metalloproteinase with thrombospondin type-1 motifs-4 [ADAMTS-4]) were assessed as the percentage of immunopositive cells in the NP and AF. With the exception of MMP-1, cytokine, and enzyme expression levels were significantly elevated in ChABC-treated discs in the NP and AF. Expression levels of TNF-α, IL1-β, and ADAMTS-4 were positively correlated with histological grade, while all cytokines and ADAMTS-4 were negatively correlated with MRI T2 and T1ρ scores. These results demonstrate that degenerate goat discs exhibit elevated expression of clinically relevant inflammatory mediators, and further validate this animal model as a platform for evaluating new therapeutic approaches for disc degeneration.     

Upregulation of CD80 on glomerular podocytes plays an important role in development of proteinuria following pig‐to‐baboon xeno‐renal transplantation – an experimental study

We have previously reported that co‐transplantation of the kidney with vascularized donor thymus from α‐1,3‐galactosyltransferase gene knockout pigs with an anti‐CD154 with rituximab‐based regimen led to improved xenograft survival in baboons with donor‐specific unresponsiveness. However, nephrotic syndrome emerged as a complication in which the glomeruli showed mild mesangial expansion with similarities to minimal change disease (MCD) in humans. Since MCD is associated with CD80 expression in glomeruli and elevated urinary excretion, we evaluated a potential role for CD80 in xenograft nephropathy. Study 1 confirmed high urinary CD80 excretion in nephrotic animals with renal xenografts showing CD80 expression in glomeruli. In Study 2, baboons receiving xenografts received CTLA4‐Ig once a week from the second postoperative week or no CTLA4‐Ig. The non‐CTLA4‐Ig group developed severe proteinuria with modest mesangial expansion with high urinary excretion of CD80 and documented CD80 expression in glomerular podocytes. All of the recipients in non‐CTLA4‐Ig groups had to be euthanized before POD 60. In contrast, CTLA4‐Ig group showed a marked reduction in proteinuria and survived significantly longer, up to 193 days. These results demonstrate that anti‐CD80 targeted therapy represents a promising strategy for reduction of proteinuria following renal xeno‐transplantation with improved survival.     

应用MB49细胞建立三种膀胱癌模型

Objective To establish three types of bladder cancer models with MB49 cells and investigate the optimal opportunity for experimental treatment. Methods The subcutaneous model of bladder cancer was established with 2×106 cells/50 uX MB49 cells;orthotopic model with 5×105 cells/50 μlMB49 cells through catheter, and pulmonary metastatic model with 1×105 cells/200 μl MB49 cells via tail vein. The optimal opportunity for experimental treatment was chosen. Results It was at the 5th day after transplantation that palpable tumor could be detected in subcutaneous model, which was the optimal opportunity for experimental treatment. The steady orthotopic model could be established by cancer cell bladder perfusion. Fist to third day was the optimal opportunity for experimental treatment. The observation duration was 3 weeks. The pulmonary metastatic model could be established by trail intravenous injection. The optimal time for experimental treatment was within 7 days. The observation duration was also 3 weeks. Conclusion The subcutaneous model, orthotopic model and pulmonary metastatic model of bladder cancer could be established by MB49 cells. The quality of the models wap good, and the time of tumor development was steady. It was favorable for choosing the optimal time of e Derimental treatment.     

应用MB49细胞建立三种膀胱癌模型

Objective To establish three types of bladder cancer models with MB49 cells and investigate the optimal opportunity for experimental treatment. Methods The subcutaneous model of bladder cancer was established with 2×106 cells/50 uX MB49 cells;orthotopic model with 5×105 cells/50 μlMB49 cells through catheter, and pulmonary metastatic model with 1×105 cells/200 μl MB49 cells via tail vein. The optimal opportunity for experimental treatment was chosen. Results It was at the 5th day after transplantation that palpable tumor could be detected in subcutaneous model, which was the optimal opportunity for experimental treatment. The steady orthotopic model could be established by cancer cell bladder perfusion. Fist to third day was the optimal opportunity for experimental treatment. The observation duration was 3 weeks. The pulmonary metastatic model could be established by trail intravenous injection. The optimal time for experimental treatment was within 7 days. The observation duration was also 3 weeks. Conclusion The subcutaneous model, orthotopic model and pulmonary metastatic model of bladder cancer could be established by MB49 cells. The quality of the models wap good, and the time of tumor development was steady. It was favorable for choosing the optimal time of e Derimental treatment.     

Sheep model for osteoporosis: Sustainability and biomechanical relevance of low turnover osteoporosis induced by hypothalamic–pituitary disconnection

Hypothalamo‐pituitary disconnection (HPD) leads to low bone turnover and osteoporosis in sheep. To determine the sustainability of bone loss and its biomechanical relevance, we studied HPD‐sheep 24 months after surgery (HPD + OVX‐24) in comparison to untreated control (Control), ovariectomized sheep (OVX), and sheep 12 months after HPD (HPD + OVX‐12). We performed histomorphometric, HR‐pQCT, and qBEI analyses, as well as biomechanical testing of all ewes studied. Twenty‐four months after HPD, histomorphometric analyses of the iliac crest showed a significant reduction of BV/TV by 60% in comparison to Control. Cortical thickness of the femora measured by HR‐pQCT did not change between 12 and 24 months after HPD but remained decreased by 30%. These structural changes were caused by a persisting depression of osteoblast and osteoclast cellular activity. Biomechanical testing of the femora showed a significant reduction of bending strength, whereas calcium content and distribution was found to be unchanged. In conclusion, HPD surgery leads to a persisting low turnover status with negative turnover balance in sheep followed by dramatic cortical and trabecular bone loss with consequent biomechanical impairment. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1067–1074, 2013