【作者】 鲜万华；

【导师】 关广聚；

【作者基本信息】 山东大学 ， 内科学， 2010， 博士

【摘要】 20世纪医学领域最辉煌的成就之一,就是器官移植成为脏器终末期疾病的治疗手段。移植的飞速发展,使移植免疫成为热点的研究领域。移植术后如何调整免疫抑制药物的方案及剂量从而使患者免疫状态的天平在排斥及感染之间达到平衡,如何减少或避免免疫抑制药物特别是钙调磷酸酶抑制剂的肾毒性作用是困扰每个肾移植临床医师的问题。目前对移植免疫学机制研究集中于体液免疫和细胞免疫,随着对移植免疫机制的认识逐渐加深,各种监测移植免疫状态的策略被应用于试验及临床。但对细胞及体液免疫的监测及认识显然仅仅还在起跑线上,现在临床上没有一种检测肾移植受者免疫状态的方法得到大家的普遍接受。目前常用的指标有：监测血清肌酐及尿素氮,应用移植肾彩色多普勒了解移植肾血流及肾动脉阻力指数,应用移植肾穿刺行组织病理活检,CSA与FK506血药浓度的检测,应用CT了解移植肾及肾窦形态,应用动态磁共振技术了解皮髓质的时间-信号强度曲线(STC)峰值等。随着移植基础及临床研究的发展,越来越多的免疫内容受到大家的关注,T淋巴细胞最早进入研究者的视野,CD4/CD8已经成为判断移植患者细胞免疫功能的代表,HLAI类、Ⅱ类抗体和MICA抗体的监测,与移植肾体液排斥反应密切相关的C4d,外周血CD15s抗原,IL-2、IL-4、IL-6、IL-10、IFN-γ、TNF-a等多种细胞因子,尿蛋白基因组学等；处于实验探索阶段的还有对Treg、调节性树突细胞和自然杀伤细胞的研究。虽然这方面的研究取得一些进展,但仍没有一种能够用于临床的非常可靠标志移植受者免疫状态的方法。临床及研究中仍以移植受者的症状体征及多种方法的联合应用,希望能准确地判断受者的免疫不足和免疫过度状态,合理地应用免疫抑制药物。第一部分探索的是肾移植巨细胞病毒感染后将他克莫司转换为环孢素时的NK及NKT细胞表达。目的以普乐可复为基础免疫抑制治疗组急性排斥反应的发生率明显低于以环孢素为基础免疫抑制治疗组,而且副作用少。因此普乐可复治疗方案在越来越多的移植中心得到广泛应用。随着肾移植数量的增多,移植并发感染的患者也越来越多,其中以巨细胞病毒(CMV)感染最为常见。以普乐可复为基础的三联免疫治疗方案中,肾移植患者中出现活动性或潜伏性CMV病毒感染后,常常减量或停用普乐可复,仅给予小剂量激素,有可能发生排斥反应,导致移植。肾功能丧失。本文评价了在CMV感染的情况下将普乐可复转换为以环孢素为基础的免疫抑制方案的安全性和免疫状态。方法通过对巨细胞病毒感染的肾移植受者随机分组,予以不同的免疫抑制方案：环孢素转换组和普乐可复减量组,进行临床观察,肾移植术后3月以上应用普乐可复(0.1～0.15mg/kg/d)+骁悉(750mmg q12h)、+类固醇激素(5mg/d)为基础的三联免疫治疗方案的患者中,活动性及潜伏性CMV感染患者共54例,按1：1随机分为2组,除抗病毒及支持对症治疗外,免疫抑制方案的调整如下：A组接受以下治疗方案：环孢素A(3～5mg/kg/d)+骁悉(0～250mgq12h)+口服或静脉应用甲强龙；B组接受以下治疗方案：普乐可复(0.05mg/kg/d+骁悉(0～250mg q12h)+口服或静脉应用甲强龙。C组为正常的肾移植患者对照组,A组患者治疗方案都采用快速转换法,立即撤除普乐可复改用环孢素,在服用环孢素5～7天后监测CsA浓度,目标浓度为100～150ng/L,如果患者在上述两种免疫方案治疗的情况下持续发热大于38摄氏度超过3天,或感染临床表现和实验室检查加重,则停用上述免疫抑制药物。主要观察的指标：在开始用药后的2、4、8周,对所有患者进行评价。动态监测人肾存活率、血肌酐、内生肌酐清除率,应用流式细胞仪检测外周血CD3+CD4+、CD3+CD8+、CD3-CD16+CD56+NK、CD3+CD16+CD56+NKT细胞的表达,应用PCR方法检测巨细胞病毒DNA定量。结果两组患者的人肾存活率无显著差异,FK506+MMF+Pred组和CSA+MMF+Pred组在治疗1～8周血药浓度分别维持在(3.1±1.2)μg/L及124±42μg/L,CsA+MMF+Pred组平均血清肌酐恢复较早,且在第2周和第4周较他克莫司组显著降低。到8周时内生肌酐清除率及在两组之间无显著差异。CD4+/CD8+和CD3-CD16+CD56+在FK506组第2周时开始出现上升,这提示了应用剂量减半的环孢素时,患者免疫抑制状态恢复较快,CD3+CD16+CD56+细胞在各时间点及各组中也存在显著差异。CsA组的巨细胞病毒定量在2周时显著低于FK506组,到第8周时两组巨细胞病毒定量均降低到10~3以下。结论肾移植患者CMV感染后除应用抗病毒及支持对症等治疗外,应用普乐可复减量及环孢素转换方案都有良好的患者和移植物存活率。使用普乐可复的肾移植受者在出现CMV感染时,转换成CsA和霉酚酸酯免疫抑制方案后,患者细胞免疫功能及肾功能恢复早,巨细胞病毒显著降低,从而降低住院时间及节约住院费用,小剂量环孢素A可能起着抑制巨细胞病毒的作用。CD3-CD16+CD56+NK细胞CD3+CD16+CD56+NKT细胞在移植肾的免疫中起着重要作用,其表达水平可预示术后感染程度的高低危状态及调整免疫抑制药物后的免疫状态据此调整免疫抑制方案,从而在防治肾移植术后巨细胞病毒感染的同时,评估CMV病毒并发排斥反应的可能性,为临床上CMV感染后免疫抑制剂的调整打下基础。第二部分探索的是CD3+CD16+CD56+自然杀伤T(NKT)细胞在外周血中的表达与移植肾排斥的关系,以及不同的血药浓度下NKT细胞的水平。目的目前对NKT细胞在肾移植中的作用及机制研究尚少。这项研究的目的就是探讨外周血NKT细胞的表达能否作为反映患者免疫状态的一个指标,根据其表达水平并结合临床来作为免疫抑制药物的调整的指南针。方法将92例肾移植受者根据血药浓度水平及有无排斥反应分为3组：正常血药浓度的无排斥组(A)、低血药浓度的无排斥组(B)、排斥组(C),10例正常人群作为对照组(D),采用流式细胞术检测各组外周血CD3+细胞、CD3+CD4+细胞、CD3+CD8+细胞、自然杀伤(NK)细胞(CD3CD16+CD56+)和NKT细胞(CD3+CD16+CD56+)及群体反应性抗体(PRA)的水平。结果正常血药浓度的无排斥组(A)、低血药浓度的无排斥组(B)排斥组(C)及对照组CD3+CD16+CD56+NKT细胞的百分数分别是(4.29±2.57)%、(4.31±2.08)%、(1.23＋1.06)%、(3.98±2.26)%；A组、B组与D组比较,NKT细胞百分数、NK细胞百分数及CD4+/CD8+无显著异常改变,PRA阳性比率无显著升高；C组NKT细胞的百分数与正常对照组相比显著降低(P<0.01),NK细胞、CD4+/CD8+均显著高于正常对照组(P<0.05),PRA阳性比率显著升高。结论肾移植受者外周血CD3+CD16+CD56+NKT细胞的表达反应了移植肾的免疫状态,当出现排斥反应时NKT细胞低表达；在无排斥反应时NKT细胞的表达稳定,而且不受低血药浓度的影响。CD3+CD16+CD56+NKT细胞在移植肾的免疫抑制中起着重要作用,根据NKT细胞的表达水平可调整免疫抑制方案,从而防治肾移植术后排斥反应的发生,减轻钙调磷酸酶抑制剂的长期肾毒性。对NK及NKT细胞在肾移植中的作用及机制研究尚少,课题设计从一定程度上为研究这一类细胞在移植免疫中的作用打开了一个新的窗口,同时也为临床上预测病人的免疫状态提供了一个新的思考方式。当移植受者测得的NK细胞、NKT细胞水平、T淋巴细胞亚群测定显示高免疫状态时,提示可能有排斥的高危险性存在；当移植受者测得的NK细胞活性或联合T淋巴细胞亚群测定显示低免疫状态时,提示可能有感染的高危险性存在,应予及时调整免疫抑制剂的种类和剂量,并应严密随访、监视。由于人体外周血淋巴细胞亚群、NK细胞、NKT细胞比例个体差异较大,系统地观察每个受者外周血CD亚群的变化规律.对肾移植受者术后感染的预防、诊断和治疗必将有很大帮助。目前的研究成果初步显示出NK及NKT细胞的检测应用于预防和监测移植排斥和感染及调整免疫方案中的重要作用。从一定程度上为研究这一类细胞在移植免疫中的作用打开了一个新的窗口。更多还原

【Abstract】 One of the 20th century’s most brilliant achievements in the medical field is to make the organ transplant as treatment for end-stage disease, With rapid development of the field in transplantation, Transplantation immunity has become a hot area of research. How to adjust immunosuppression programs and the drug dose after transplantation to the immune status in the balance between rejection and infection, how to reduce or avoid renal toxicity of the immunosuppressive drugs, especially calcineurin inhibitor is a problem to each a kidney transplant clinicians. Current mechanism of transplantation immunology focused on the humoral and cellular immunity, with the knowledge of the mechanisms of transplantation immunity gradually deepened, a variety of strategies to monitor the state of transplantation immunity has been used in experimental and clinical. But the cellular and humoral immunity is clearly still in the starting line only, and now no one clinical test of renal transplant recipients is universal acceptance to evaluate immune status.The commonly used indicators are:monitoring of serum creatinine and blood urea nitrogen, application of color Doppler to understand the shape of graft、renal artery resistance index、renal blood flow, application of CT to understand renal sinus and renal morphology, using dynamic magnetic resonance imaging to understand corticomedullary time-signal intensity curve (STC) peak, application of renal puncture biopsy, CSA and FK506 blood concentration testing. With the development of experimental and clinical research, more and more immunologic content is concerned about。T cells were the first entering the field of researchers, CD4/CD8 cell have become an indicator of immune in transplant patients, Treg, regulatory dendritic cells and natural killer cells was also in the experimental stage of exploration. Humoral rejection such as HLAⅠclasses,ⅡClass antibodies and MICA antibody monitoring, and C4d, blood CD 15s antigen, IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-αand other Cytokines, urinary protein genomics are also in exploration. Although these research have made some progress, but still no one of them can be thought a very reliable marker to evaluate the immune status for clinical transplant recipients. The clinical signs、symptoms and a variety of methods are combined and applied in transplant recipients, hoping to accurately determine the deficiency and excessive of immune status in the recipients, and hoping to a reasonable application of immunosuppressive drugs.The first part explores the expression of NK and NKT cells in the kidney transplant patients after cytomegalovirus infection with adversing tacrolimus-based immunosuppressive therapy to cyclosporin.Objective The incidence of rejection in tacrolimus-based immunosuppressive therapy group was significantly lower than that in cyclosporine-based immunosuppressive therapy group, and with few side effects. Therefore, tacrolimus treatment is widely used in more and more transplant center. With the increase of renal transplantation, complicated infections such as cytomegalovirus (CMV) infection in transplant patients become common. When patients with tacrolimus-based immunosuppressive therapy appear active or latent CMV infection, tacrolimus always is reduced or unused, or steroids is only used, Rejection may happen, resulting in loss of graft function. Safety and immune status will be evaluated converting tacrolimus to cyclosporine-based immunosuppressive in these patients with CMV infection.Method Patients after renal transplantation more than 3 months were treated with triple immunosuppressive treatment of tacrolimus (0.1-0.15mg/kg/d)+ mycophenolate mofetil MMF (750mg q12h)+steroids (5mg/d), and were infected with active or latent CMV infection,54 patients were randomLy divided into 2 groups according to 1:1, in addition to supported for anti-virus and symptomatic treatment, the immunosuppressive treatment were adjusted as follows:group A:CsA adversion group, cyclosporine A (3-5mg/kg/d)+mycophenolate mofetil MMF (0-250mg q12h)+oral or intravenous methylprednisolone; Patients were treatment with fast conversion method, immediately conversion tacrolimus to cyclosporine. concentration of cyclosporine was monitored 5-7 days after taking CsA, target concentration was 100-150ng/L.group B:tacrolimus reduction group,tacrolimus (0-0.05mg/kg/d)+MMF (0-250mg ql2h)+oral or intravenous methylprednisolone. If patients in these treatments persist fever over 38 degrees more than 3 days, or infect increase with the clinical and laboratory tests, then remove the immunosuppressive drugs of CNI and MMF.Indicators were observated:at the beginning of the 4,8 weeks after treatment, all patients were evaluated.monitoring of serum creatinine, creatinine clearance rate, the incidence of acute rejection, mortality and graft loss rate, pulmonary infection, monitoring blood CD3+CD4+, CD3+CD8+, CD3-CD16+CD56+ NK, CD3+CD16+CD56+ NKT cells and CMV DNA quantitativeResults Two groups of patients were no significant differences in graft survival and mortality rate, FK506 decrese group and the CSA adverse group of 1-8 weeks in the treatment of blood concentrations were maintained at (3.1±1.2)μg/L and 124±42ug/L, These showed that the average serum creatinine in CsA group,recovery earlier than in FK506 group. But it was not significantly different between two groups at week 8. CD4/CD8 and CD3-CD16 CD56 in the CsA group the first 2 weeks up to 4 weeks and 8 weeks to reach a plateau curve, CD4/CD8 and CD3-CD16 CD56 in the FK506 group increased when the first 2 weeks, to 8 weeks to the peak value, which suggested that the patient recovered rapidly from immunosuppression state when half dose of cyclosporine was applied. CD3 CD16 CD56 cells and the group at all time points, there are also significant differences like CD3-CD16+CD56+ cells. At weeks 2 Cytomegalovirus quantitatively in CsA group was significantly lower than in FK506 group, to weeks 8 CMV quantitative in two groups were reduced to less than103 copy per mL.Conclusion Patients with CMV infection after renal transplantation were treated with anti-virus and other symptomatic treatment, application of tacrolimus reduction and cyclosporine conversion programs have good patient and graft survival. Use of tacrolimus in renal transplant recipients in the event of CMV infection, converted into CsA and mycophenolate mofetil immunosuppressive regimen, the cellular immune recoved earlier, Cytomegalovirus was significantly reduced earlier in weeks 2, thus reduced hospital stay and save hospital costs. CD3-CD16+CD56+Nkcells and CD3+CD16+CD56+NKT cells in the transplanted kidney plays an important role in immune. Its expression can predict the extent of infection before and after adjustment immunosuppressive drugs. Its expression can provide a clue for Immune status in prevention and treatment of cytomegalovirus infection at the same time, assess the possibility of CMV virus combination with rejecti o.The second part explores CD3+CD16+CD56+natural killer T (NKT) cells in the peripheral blood in graft rejection and in different blood concentrations.Objective NK and NKT cells present in renal transplantation and its mechanism is still unkwown. This article discusses CD3+CD16+CD56+ natural killer T (NKT) cells in the peripheral blood between different immuno state and different blood concentrations. The purpose of this study is to explore the peripheral blood NK and NKT cells can reflect the immune status as an indicator, combination with clinical expression. According to NKT cells immunosuppressive drugs can be adjusted.Methods 92 patients after renal transplantation were divided into three groups according to rejection and the concentration of calcineurin inhibitor: patients without rejection and within normal concentration range (group A), patients without rejection and within lower concentration range (group B), patients with rejection (group C),10 healthy individuals were selected as controls. The proportion of CD3+、CD3+CD4+、CD3+CD8+、CD3-CD16+CD56+ NK CD3+CD16+CD56+ NKT cells and panel reactive antibody(PRA) were analyzed using flow cytometry.Results The proportion of NKT cells in the patients of group A, group B, group C, and group D were (4.29±3.57)%、(4.31±3.08)%、(1.23±1.06)%、(3.98±2.26)%,respectively. The proportion of NKT、NK cells、CD4+/CD8+、PRA in group A and B did not change significantly compared to those in group D.The proportion of CD3+CD16+CD56+ NKT cells in the peripheral blood of patients was lower in group C compared with that in group D, Significant increase in NKT、NK、CD4+/CD8+、PRA were observed in group CConclusions Monitoring of the expression of CD3+CD16+CD56+ NKT cells in peripheral lymphocytes after renal transplantation may contribute to immune Status. Lower proportion of the CD3+CD16+CD56+ NKT cells indicate rejection. The expression of NKT cells were not affected by lower concentration in patients without rejection. Natural killer T (NKT) cells play an important role in renal transplantation. Analysis of NKT cells can identify patients at risk of graft rejection, and can allow minimization of calcineurin inhibitor and avoidance of its side effects.NK and NKT cells in kidney transplantation and their mechanism are still unkown,To some extent,project design of this study provides a new way of thinking for predicting the clinical immune status of patients.when NK cells, NKT cell level, T lymphocyte subsets tests showed high immune status in the transplant recipients, suggesting that there may exist high risk of rejection; When They showed low immune status, suggesting that the high risk of infection, The program and dose of immunosuppressive drugs should be adjusted. Since human peripheral blood lymphocyte subsets, NK cells, NKT cells are individual differences in a larger proportion, systematic observation of each subsets of peripheral blood CD variation should be does. Postoperative infection in renal transplant recipients for prevention, diagnosis and treatment will be of great help. Current study shows that NK and NKT cells play a important role when used in prevention and detection of transplant rejection and infection,used in monitoring and adjustment the immunization state. This type of cell in the role of transplantation immunity opens a new window, provides a new way of thinking for predicting the clinical status of the patient’s immune.更多还原