【作者】 王仁定；

【导师】 陈江华；

【作者基本信息】 浙江大学 ， 外科学， 2002， 硕士

【摘要】 1．引言 新型免疫抑制剂的应用明显降低了肾移植术后急性排斥的发生，改善了短期效果，但人／肾的存活并没有同步提高。并且长期应用免疫抑制剂带来许多严重的副作用，包括肾毒性，增加机会感染，加速动脉硬化以及增加恶性肿瘤的发生等。因此，不应用免疫抑制剂而提高人／肾的存活，诱导移植免疫耐受是移植界共同的理想。 目前，诱导移植免疫耐受方法很多，包括放疗+供体骨髓植入，供体特异性输血，应用单克隆抗体，供体特异性骨髓输注等，而肾移植术后供体特异性骨髓细胞(Donor-specific Bone Marrow Cells，DBMC)输注诱导供体部分特异性免疫耐受是被认为比较现实的措施，具有较大的临床应用前景。虽然，DBMC诱导免疫耐受机制不甚清楚，但自1993年Starzl提出细胞移行与嵌合体是移植物被接受并长期存活的基础以来，不少学者认为DBMC诱导免疫耐受可能与其促进嵌合体的产生和维持有关。 迈阿密研究中心最近的系列研究提示DBMC输注可以提高人／肾的存活，促进嵌合体的形成，降低急慢性排斥，而嵌合体的形成在诱导和维持免 2002年浙江大学硕士学位论文 疫耐受中起重要的作用。国内单中心对未进行DBMC输注肾移植受者外周 血嵌合体检测，结果提示嵌合体随着时间推移，其出现的频率是增加的。 在国内，由于缺乏操作性良灯的DBMC获取技术，限制了有关DBMC 输注与移植免疫耐受的研究。自1999年1月起，浙江大学附属第一医院肾 脏病中心建立了具有较强可操作性的供体椎体骨髓的获取技术，率先开展肾 移植后供体特异性骨髓输注的前瞻性临床对照研究，至2001年12 H共完成 63例受者的供体特异性骨髓输注，同期未进行DBblC输注病例425例。同时 发现DBMC输注是安全的，对嵌合体的定性检测提示DBMC输注早期有促进嵌 合体形成的作用。 目前国内外检测嵌合体的方法主要为定性方法包括本中心己建立 PCRSSP和PCR．SRY法，由于不能量化分析，易受各种因素的影响，且分 辨率不高，因此，本课题拟建立定量检测嵌合体的PCR－FIOW方法，对肾移 植受体外周血单个核细胞嵌合体的存在进行检测：并通过对浙一肾脏病中心 DB＼IC输注病例的嵌合体检测及对照组的比较，试图了解在国人中，按浙一 肾脏病中心的摸式进行DBMC输注，DBMC对肾功能、急性排斥及嵌合体的影 响，KVh存活术进行DBMC M例中嵌合体的惰况。 2．实验方法 2＊实验检测部分 排除肾移植术前接受外周血输注，未保存供、受者木前淋巴细胞标本， 术后多个时间点未留取外周血淋巳细胞标本及供受者HLA—DR位点分型 完全相同的病例，共选择肾移植术后联合DBMC输注病例36例（DBMC 组）、同期配对病例中选取26例（配对对照组）。5年以上长期存活女性病 例15例（长期病例组）。对上述各组在相应时间点（DBMC组及配对对照 织：术前、术后1周、术后4周、术后8周、术后22－26周、术后4452周、 3 2002年浙江人学呸I：学位论义 术后52周以卜；卜期病例组：门诊随认时）留取外周血淋巴细胞标本，经 DNA抽提、供受体HLA－DR位点确定后，利用供体特异性位点通过 PCR．FI。W方法检测供体细胞。模拟不同比例的嵌合体以确定方法的敏感性， 并利用普通PCR扩增、产物电泳、凝胶成像和分析，以确定方法的特异性。 2二临床部分 1999年1月至2001年12月，本中心共进行尸体肾移植488例，排除 多器官联合移植，二次移植，早期处亡门月内），延迟复功等后469例。观 察组63例，其中男性44例，女性19例平均年龄45．3岁（3－64岁），术后 免疫抑制剂方案为：环抱素＋骁悉＋强的松（C＋M＋P）54例，普乐可复＋骁悉＋ 强的松厂＋M＋P）9例，供体骨髓分3次输入，总量为币l士厂l）X＊／kg。 对照组 406例，其中男性 271例，女性 135例，平均年龄 39．8岁（15－70岁）， 术后免疫抑制方案为：C＋M＋P 364例，F＋M＋P 42例。两组均不应用抗淋巳 细胞球蛋白制剂诱导治疗。 3．实验结果 3＊实验检测部分 31j各织间基本情况的分析 通过F方更多还原

【Abstract】 1.IntroductionNew potent immunosuppressive drugs have significantly decreased acuterejection, improved short-term effects of renal allograft, but without improvement of recipient/allograft survival. What’s more, these drugs are associated with many severe side effects, including nephrotoxicity, susceptibility to opportunistic infections, accelerated atherosclerosis, and malignancy. As a result, the "induction" of donor specific tolerance has been one of the primary goals of researchers in the field of transplantation immunology.Until now, several strategies were used to induce donor specific tolerance, but the realizable way to induce at least some forms of operational tolerance is donor-specific bone marrow (DBMC) infusion. However, the mechanism of DBMC infusion is not clear, in 1993, Starzl proposed that organ allograft acceptance was associated with the survival of bone marrow-derived stem cells, which was assimilated into the larger immunological network of the host, lots of experts consider it’s associated with augmentation and maintenance of chimerism. Under such circumstance, recipients can benefit from low dosage ofimmunosuppressive agents, less adverse effects, longer survival of allografts.Until recently, a series of studies in Miami demonstrated that DBMC infusion can significantly increase recipient and allograft survival, decrease acute/chronic rejection, they found that chimerism plays crucial role in inducing and maintaining hyporesponsiveness or unresponsiveness. In our country, one centers showed that chimerism exists in transplant recipients, and frequency of chimerism increaseith time.In order to explore the regulatory mechanisms involved and to follow the clinical outcome in the hope of eventually reducing the burden of generalized immunosuppression, a series of kidney transplant recipients, post-operatively infused with DBMC in the absence of a bone marrow ablation protocol, have been followed at our center since January 1999. The present study describes the 3-year actuarial patient and graft survival of a series of 63 first cadaver kidney transplant recipients (DBMC group) who received transplants between January 1999 and December 2001, and were given donor vertebral body bone marrow and prospectively followed. Comparing with 425 renal allografts at the same time interval, they were not given marrow treated with an equivalentimmunosuppressive regiment, and followed prospectively. We found DBMCi/’fcuW’ infusion is safe and augmention of chimerism can be seen.The objective of this study is to establish PCR-Flow assay to quantitate microchimerism, and try to affirm the effect of DBMC infusion to augment the level of microchimerism, to confirm the correlation between DBMC transfusion and acute rejection, and so on.2.Methods2.1 Experiment partAccording to the accepting and eliminating standards, 77 recipients werescreening out, 36 of them accepted DBMC infusion after cadaver renaltransplantation (DBMC group). 26 of them belong to the control group, and 15of them belong to the long-term survival group who live with the good functional allograft more than 5 years.Peripheral blood mononuclear cells (PBMC) were obtained using Ficoll-Hypaque density gradient centrifugation on schedule (1 week, 4weeks, Sweeks, 22-26weeks, 44-52weeks, over 52weeks for DBMC and control group postoperatively). Then through the extract of genomic DNA, affirmance of the donor and recipients HLA-DR alleles, PCR-Flow assay to detect fluoresceinated peripheral blood mononuclear cells (chimerism or microchimerism).2.2 Clinical partFrom January 1999 to December 2001, excluding multi-organs transplant, second transplant, early death (within one month), delayed graft function, there were 63 patients (observed group) who infused with donor vertebral body bone marrow cells for three times postoperative!}, 406 patients (control group) who were not infused with DBMC at the same interval. Immunosuppression consisted of prednisolone + mycophenolate更多还原