供体脾灌注对高度致敏患者移植肾的免疫保护作用

目的探讨供体脾灌注对高度致敏患者移植肾的保护作用及机理。方法对16例高度致敏患者进行配对分组,实验组肾移植术中先予供体脾灌注。前瞻性观察脾灌注对受者群体反应性抗体（penel reactive antibody,PRA）、补体依赖细胞毒性（complement dependent cytotoxicity,CDC）试验、血清补体溶血活性（CH50）、可溶性人类白细胞抗原Ⅰ（soluble human leucocyte antigen-Ⅰ,sHLA-Ⅰ）以及术后早期移植肾排斥反应和移植肾功能的影响。结果供体脾灌注40min后,患者的PRA、CDC和CH50较灌注前显著降低,血清sHLA-Ⅰ浓度显著增加。对照组肾移植术后发生超急性排斥1例,加速性排斥反应3例,急性排斥反应3例。对照组排斥反应严重程度明显较灌注组高（u=2.54,P〈0.05）;对照组排斥反应发生时间为（3.3±2.6）d（中位数3.0d）,脾灌注组为（10.5±6.2）d（中位数8.5d）,组间比较差异具有统计学意义（t=10.46,P〈0.01）;术后近期血肌酐（serumc reatinine,Scr）值显著高于脾灌注组。结论供体脾灌注可以延缓高度致敏患者术后近期移植肾排斥反应的发生,减轻排斥反应强度,改善移植肾功能;其机制可能与供体脾脏特异性吸附受者毒性抗体,消耗血清补体和分泌s HLA-Ⅰ类抗原有关。     

受者外周血淋巴细胞黏附分子基因表达对肾移植急性排斥反应的预测作用

目的 探讨肾移植受者外周血淋巴细胞LFA-1、ICAM-1基因表达水平对术后急性排斥反应的预测作用.方法 (1)在取肾同时取供体脾脏,并制成脾细胞悬液.在术前及术后第4天分别抽取受者外周静脉血,并分离出淋巴细胞.同时跟踪调查受者术后1个月内急性排斥反应的发生情况.(2)以供者及无关者脾细胞为刺激细胞,受者淋巴细胞为反应细胞,做混合淋巴细胞培养.采用MTT法测定供者及无关者脾细胞刺激受者淋巴细胞增殖的刺激指数.采用半定量RT-PCR方法测定在供体脾细胞、无关者脾细胞刺激及未刺激时,受者淋巴细胞LFA-1、ICAM-1基因表达水平.结果 (1)本组研究了35例能够获取外周血淋巴细胞及供体脾细胞的患者,术后1个月内发生过急性排斥反应10例,未发生急性排斥反应25例,急性排斥反应发生率28.6%.(2)术前及术后第4天时,供体及无关者脾细胞刺激受者淋巴细胞增殖的刺激指数在发生急性排斥反应与未发生急性排斥反应受者之间差异无统计学意义(P＞0.05).术后第4天,正常组淋巴细胞对供体脾细胞反应的刺激指数术后较术前的下降值明显高于排斥组,二者之间差异有统计学意义(P＜0.05);而正常组和排斥组淋巴细胞对无关者脾细胞反应的刺激指数术后较术前的下降值差异无统计学意义(P＞0.05).(3)术前及术后4d,未刺激时,受者淋巴细胞LFA-1、ICAM-1基因表达水平在发生急性排斥反应与未发生急性排斥反应受者之间差异无统计学意义(P＞0.05);术前及术后4d,无关者脾细胞对受者淋巴细胞刺激时,受者淋巴细胞LFA-1、ICAM-1基因表达水平在发生急性排斥反应与未发生急性排斥反应受者之间差异无统计学意义(P＞0.05);术前及术后4d,供者脾细胞对受者淋巴细胞刺激时,受者淋巴细胞LFA-1、ICAM-1基因表达水平在发生急性排斥反应与未发生急性排斥反应的受者之间差异有统计学意义(P＜0.01).结论 (1)术前、术后受者淋巴细胞对供体特异抗原及无关者抗原反应的刺激指数,对术后急性排斥反应发生无预测作用;而术后,受者淋巴细胞对供体抗原反应的刺激指数较术前的下降值可能有一定的预测作用.(2)术前、术后在无关者抗原刺激及无抗原刺激时,肾移植受者外周血淋巴细胞LFA-1、ICAM-1基因表达水平对术后急性排斥反应发生无预测作用.(3)术前、术后在供体特异抗原刺激时,肾移植受者外周血淋巴细胞LFA-1、ICAM-1基因表达水平对术后急性排斥反应发生有明显预测作用.     

HLA体液致敏与肾移植

目的：探讨肾移植受者HLA体液致敏的形成机制及其对肾移植术后排斥反应发生的影响，同时评价其对排斥反应的辅助诊断作用。方法：采用ELISA方法对199例次肾移植受者定期进行HLA抗体水平检测，分析其与患者的年龄、性别、透析时间、移植史、输血次数、孕次、冷缺血时间、热缺血时间、HLA体液致敏及HLA错配数、术后早期排斥反应的发生与恢复、肾功能延迟恢复的关系。结果：单因素分析显示性别、移植史、输血、妊娠、透析时间与HLA致敏相关（P＜0．05），致敏及HLA错配与排斥反应发生相关（P＜0．05）；而多因素分析提示移植史、输血与致敏相关，致敏及HLA错配与排斥反应发生相关，而且致敏与肾功能延迟恢复发生相关。HLA抗体水平升高和减低与排斥反应的发生和逆转呈正相关。结论：移植史、输血、妊娠和长期透析是移植受者HLA致敏的重要因素；致敏及HLA错配是排斥反应及肾功能延迟恢复的主要危险因素；HLA抗体水平检测对排斥反应的发生和逆转有辅助诊断作用。     

供体特异性输血在亲体肾移植的初步分析

目的 探讨供体特异性输血对亲体肾移植患者围手术期并发症和术后人肾存活率的影响.方法 将我院2005-2007年收治的52例亲体肾移植患者按是否进行供体特异性输血分为供体输血组和对照组,供体输血组于术前3 d采集全血200～400 ml,术中用于受者;对照组术中随机使用库存血.对比分析两实验组在肾移植后移植肾的功能(血肌酐测定)、急性排斥反应发生率、移植肾功能延迟恢复(delayed graft function,DGF)发生率.结果 供体输血组和对照组在供肾GFR、HLA配型、供者和受者年龄方面无显著差异(P＞0.05).供体输血组术中输供体全血(334±56.4)ml,随机输注浓缩红细胞(238±105)ml;对照组输注浓缩红细胞(698±243)ml.供体输血组与对照组术后1周血肌酐水平无显著差异(P＞0.05),术后急性排斥反应和DGF发生率低于对照组(分别为3.57%、16.67%).结论 供体特异性输血可明显减少移植术中库存非亲体血使用量,减少术后急性排斥和DGF发生率,有效提高了移植受者围手术期的成功率.     

肾移植后外周血单个核细胞中Foxp3 mRNA的表达

目的:研究外周血单个核细胞中Foxp3 mRNA表达水平与同种异体肾移植术后急性排斥反应及慢性移植肾肾病发生的关系。方法:应用实时荧光定量聚合酶链式反应（RT-PCR)的方法检测16例肾移植术后急性排斥患者、8例慢性移植肾肾病患者、8例移植后肾功能正常者、8例尿毒症患者和8例正常人的新鲜外周血单个核细胞中Foxp3 mRNA的表达水平,比较各组的差异及其与急性排斥反应和慢性移植肾肾病发生的关系。结果:急性排斥反应患者外周血中Foxp3 mRNA表达水平低于慢性移植肾肾病患者,差异有统计学意义（P<0.01);慢性移植肾肾病患者外周血中Foxp3 mRNA表达水平明显低于正常人、终末期肾病患者和肾移植后肾功能正常患者,差异有统计学意义（P<0.01);正常人、终末期肾病患者和肾移植后肾功能正常患者之组间Foxp3 mRNA表达水平无显著差异（P>0.05)。结论:外周血中Foxp3 mRNA表达水平可以反应移植肾的功能状态,其可能成为早期诊断移植后急性排斥反应和慢性移植肾肾病的一个无创指标。     

同供体肾移植及骨髓细胞输注诱导免疫耐受临床研究初步报告

目的：通过同供体肾移植及骨髓细胞输注，观察受者体内微嵌合体形成情况，探索同供体肾移植及骨髓细胞输注的安全性及近期效果。方法：4例肾移植受者于肾移植术后第1天、第3天输注同供体骨髓细胞，并与接受同一供体肾移植患者进行移植后的临床效果观察及免疫状态的分析比较。结果：接受同供体骨髓输注的4例肾移植受者临床效果良好，均产生免疫低反应。结论：同供体肾移植及骨髓细胞输注是一种可行的诱导免疫耐受的方法，且简便安全。     

亲属活体肾移植48例报告

目的 探讨活体肾移植的供受者选择标准、治疗方法和随访结果.方法 分析1998年3月至2008年7月我院实施的48例亲属活体肾移植的供受者临床资料、评估、配型、手术方法及免疫抑制剂方案.结果 全部供者术后未出现并发症,术后肾功能正常.48例受者中有不同程度的排斥反应、移植肾功能延迟恢复、感染等并发症.结论 亲属活体肾移植排斥反应明显低于尸体肾移植,具有经济、移植肾长期存活效果好的优势,但同时也存在一些伦理学的争议.     

应用流式补体依赖淋巴细胞毒技术检测肾移植病人HLA抗体

目的 为了更好地识别造成移植肾排斥的特异性抗供者HLA的IgG类型同种抗体,对Flow-CDC方法应用于肾移植及其临床相关性进行研究.方法 对96例等候肾移植受者同时进行PRA、NIH-CDC和Flow-CDC实验,并观察其中34例接受NIH-CDC阴性肾移植术的受者近期移植效果. 结果 Flow-CDC和NIH-CDC两种实验方法的阳性率[27.8%(42/151)和17.3%(26/151)]之间差异有统计学意义(χ2=4.86, P<0.05).另外, PRA阴性受者其NIH-CDC和Flow-CDC均为阴性,阴性吻合率为100%;在接受同种异体肾移植术的34例受者,其中20例PRA阴性受者接受了NIH-CDC和Flow-CDC均阴性供肾,移植后未发生排斥,移植肾功能迅速恢复;13例PRA阳性的致敏受者接受NIH-CDC和Flow-CDC均阴性供肾的,1例发生急性排斥经治疗后逆转,12例无排斥移植肾功能良好;1例PRA阳性再次移植受者接受了NIH-CDC阴性而Flow-CDC阳性的供肾,移植后第2天出现少尿,10d切除移植肾.结论 Flow-CDC方法是一种能够识别具有补体结合能力的抗供者特异性HLA抗体的交叉配型技术,比经典的NIH-CDC方法具有更敏感、可标准化、快速等优点,对预示肾移植术后的排斥反应方面更具有前瞻性.     

PRA配型技术在致敏受者肾移植术中的应用研究

目的 探讨群体反应性抗体(PRA)配型技术在致敏受者肾移植中的临床效果.方法 应用抗原板(LAT),采用酶联免疫吸附法(ELISA)检测肾移植受者术前的PRA;采用PRA配型技术进行术前配型.结果 12例致敏受者组采用PRA配型技术,肾移植术后肾功能恢复正常,无1例发生超急性排斥反应,术后1个月内急性排斥反应的发生率为25%;同期43例非致敏受者组,术后1个月内急性排斥反应的发生率为18.6%,虽较致敏受者组低,但两组之间差异无统计学意义.结论 PRA配型技术对减少致敏受者肾移植排斥反应,提高移植物存活率具有重要意义.     

犬肾移植致敏模型的建立

目的建立犬致敏后的肾移植动物模型。方法雄性家犬各6条配对作为淋巴细胞供受体,采用小剂量（0.4×10^7-1.2×10^7个/kg）淋巴细胞多部位多次输注的方法诱导致敏。用补体依赖的淋巴细胞毒（complement deendent cytotoxicity,CDC）和混合淋巴细胞培养（mixed lymphocyte culture,MLC）试验进行检测。当CDC转为阳性和MLC显示反应淋巴细胞增殖明显活跃后,淋巴细胞供受体犬间进行交叉肾移植,术后定期ECT动态显像观察移植肾功能变化;分批摘取移植肾行病理检查,观察排斥反应发生情况。结果实验犬均在输注淋巴细胞3-4次后CDC转为阳性,MLC显示反应淋巴细胞增殖明显活跃。肾移植术后4天ECT检查有4条致敏犬移植肾血流灌注下降,肾功能出现损害,摘取的3只移植肾病理检查表现为抗体介导的排斥（延缓）或急性排斥反应;对照组未见异常。术后7天,致敏犬移植肾均因急性排斥失功;对照组3条犬有2条出现肾功能损害,病理检查示急性排斥反应。结论小剂量淋巴细胞多部位多次输注可以诱导出家犬的免疫致敏状态。致敏犬肾移植术后移植肾排斥反应出现较早,肾功能损害更严重。     

Establishment of a sensitized canine model for kidney transplantation

Objective:To establish a sensitized canine model for kidney transplantation. Methods: 12 male dogs were averagely grouped as donors and recipients. A small number of donor canine lymphocytes was infused into different anatomic locations of a paired canine recipient for each time and which was repeated weekly. Specific immune sensitization was monitored by means of Complement Dependent Cytotoxicity (CDC) and Mixed Lymphocyte Culture (MLC) test. When CDC test conversed to be positive and MLC test showed a significant proliferation of reactive lymphocytes of canine recipients, the right kidneys of the paired dogs were excised and transplanted to each other concurrently. Injury of renal allograft function was scheduled determined by ECT dynamic kidney photography and pathologic investigation. Results:CDC test usually conversed to be positive and reactive lymphocytes of canine recipients were also observed to be proliferated significantly in MLC test after 3 to 4 times of canine donor lymphocyte infusions. Renal allograft function deterioration occurred 4 d post-operatively in 4 of 6 canine recipients, in contrast to none in control dogs. Pathologic changes suggested antibody-mediated rejection (delayed) or acute rejection in 3 excised renal allograft of sensitized dogs. Seven days after operation, all sensitized dogs had lost graft function, pathologic changes of which showed that the renal allografts were seriously rejected. 2 of 3 dogs in control group were also acutely rejected. Conclusion：A convenient method by means of repeated stimulation of canine lymphocyte may induce specific immune sensitization in canine recipients. Renal allografts in sensitized dogs will be earlier rejected and result in a more deteriorated graft function.     

Use of PCR and PCR-SSP for detection of urinary donor-origin DNA in renal transplant recipients with acute rejection

Objective To analyze the urine of renal recipients for the pressence of donor DNA in an attempt to establish an alternative diagnostic means of acute rejection.Methods Sixty-four renal transplant recipients were examined.Thirty-seven were normal after transplantation,while 22 others developed acute rejection,based on serum creatinine levels and/or needle biopsy findings of the graft.Five developed drug-induced renal dysfunction.In female recipients with a male graft,we examined urine for the presence of Ychromosome(SRY and DYZ-1) and in recipients receiving and HLA mismatched graft,we looked for HLA-DR gene(DRB1)using PCR.Results Among the 14 female recipients with male grafte demonstrating stable renal function,only one was positive for SRY and DYZ-1 on the Y chromosome.However,sry AND DYZ-1 were found in the urine of four female patients with acute rejection,but these DNA fragments were not detected in 3 of the 4 after anti-rejection therapy.The last patient was referred to hemodialysis.Of 23 recipients of a graft from HLA mismatch donors with stable renal function,DRB1 was negative in 21(91%).Of 18 patients with acute rejection,DRB1 was positive in 16(89%)and negative in 2.these ENA fragments were no longer found in 13 patients after anti-rejection therapy.In all patients with drug induced renal dysfunction,donor-derived DNA was negative.Conclusions Presence of door specific DNA in the urine of the recipient is strongly associated with acute rejection.Analysis of dna DNA derived from donor cells in urine was an effective and accurate method for the diagnosis of acute rejection of a renal transplant.     

Rituximab induction therapy in highly sensitized kidney transplant recipients

Background  The number of highly sensitized patients is rising, and sensitization can lead to renal transplant failure. The present study aimed to investigate the safety and efficacy of renal transplantation following induction therapy with rituximab in highly sensitized kidney transplant recipients.

Methods  Seven highly sensitized kidney transplant recipients who underwent rituximab therapy from December 2008 to December 2009 were retrospectively analyzed. There were 3 men and 4 women, with a mean age of 38.5 years (range, 21–47 years). The duration of hemodialysis was 3–12 months, with a mean duration of 11 months. For 4 patients, this was the second transplant; the previous graft survival time was 2–11 years, with a mean survival time of 5.8 years. All the female recipients had history of multiple pregnancies, and all patients had previously received blood transfusions. All donors were men, with a mean age of 32.5 years (range, 25–37 years). In 2 of the 7 patients, both class I and class II of panel reactive antibody were high; the remaining 5 patients showed either high in class I or in class II of panel reactive antibody. The mean panel reactive antibody value was 31% for class I and 51% for class II respectively. The donors and the recipients had the same blood type, with low lymphocyte cytotoxicity ranging from 2% to 5%. The human leukocyte antigen (HLA) mismatch numbers were from 2 to 4. All patients received tacrolimus (0.1 mg∙kg^-1∙d^-1) and mycophenolate mofetil (750 mg twice per day) orally 3 days prior to surgery. All patients received a single dose of 600 mg rituximab (375 mg/m²) infusion on the day before surgery and polyclonal antibody (antithymocyte globulin) on the day of surgery. Postoperative creatinine, creatinine clearance rate, and occurrence of rejection by pathological biopsy confirmation were monitored.

Results  No patient had delayed graft function after surgery. Two patients had acute rejection, one on day 7 and the other on day 13 post-surgery. Diagnosis of acute rejections was based on the clinical assessments and pathological biopsy results. According to the Banff 07 classification of renal allograft pathology, one of the patients was Ia and the other was IIa; the C4d staining was negative in both patients. One patient received methylprednisolone plus cyclophosphamide and the other received antithymocyte globulin (ATG) therapy, both leading to successful reversion of the acute rejection. All patients were discharged postoperatively and all had normal renal function during the 7th to 12th month follow-up. Pulmonary infection occurred in 1 patient 4 months after surgery and was successfully cured.

Conclusion  Rituximab induction therapy can reduce the occurrence of postoperative humoral rejection in highly sensitized renal transplant recipients, suggesting that kidney transplantation may be safe and effective for these patients.

     

Coexistence of Th1/Th2 and Th17/Treg imbalances in patients with allergic asthma

Background  Recent recognition is that Th2 response is insufficient to fully explain the aetiology of asthma. Other CD4⁺ T cells subsets might play a role in asthma. We investigated the relative abundance and activities of Th1, Th2, Th17 and CD4⁺CD25⁺ Treg cells in patients with allergic asthma.

Methods  Twenty-two patients with mild asthma, 17 patients with moderate to severe asthma and 20 healthy donors were enrolled. All patients were allergic to house dust mites. Plasma total IgE, pulmonary function and Asthma Control Questionnaire were assessed. The proportions of peripheral blood Th1, Th2, Th17 and CD4⁺CD25⁺ Treg cells were determined by flow cytometry. The expression of cytokines in plasma and in the culture supernatant of peripheral blood mononuclear cells was determined by enzyme linked, immunosorbent assay.

Results  The frequency of blood Th2 cells and IL-4 levels in plasma and culture supernatant of peripheral blood mononuclear cells were increased in all patients with allergic asthma. The frequency of Th17 cells and the plasma and culture supernatant levels of IL-17 were increased, whereas the frequency of CD4⁺CD25⁺ Treg cells and plasma IL-10 levels were decreased in patients with moderate to severe asthma. Dermatophagoides pteronyssinus specific IgE levels were positively correlated with the percentage of blood Th2 cells and plasma IL-4 levels. Forced expiratory volume in the first second was negatively correlated with the frequency of Th17 cells and plasma IL-17 levels, and positively correlated with the frequency of Treg cells. However, mean Asthma Control Questionnaire scores were positively correlated with the frequency of Th17 cells and plasma IL-17 levels, and negatively correlated with the frequency of Treg cells.

Conclusions  Imbalances in Th1/Th2 and Th17/Treg were found in patients with allergic asthma. Furthermore, elevated Th17 cell responses, the absence of Tregs and an imbalance in Th17/Treg levels were associated with moderate to severe asthma. 

     

影响致敏患者移植肾存活的危险因素分析

目的探讨影响致敏患者移植肾存活的危险因素,识别引起移植物失功的高危患者,以提高致敏患者移植肾长期存活率。方法选择102例行肾移植术的致敏患者进行回顾性研究,用Kaplan-Meier计算1、3、5年移植肾存活率,用log-rank进行单因素分析和Cox模型多因素回归分析,计算相对危险度。结果102例致敏患者随访期间移植肾失功16例,其中死亡7例,术后1年内死亡5例,术后2及3年带肾死亡各1例。死亡原因肺部感染5例、心血管疾病2例,失访3例。1、3、5年人存活率为95%、93%和93%,1、3、5年肾存活率为90%、85%和75%,移植肾半生存期为8.9年。单因素及多因素分析表明受者年龄、移植次数、PRA水平、术后PRA水平升高、HLA相配程度、移植肾功能恢复正常时间、移植肾功能延迟恢复、急性排斥反应、血肌酐水平、感染等10个因素对移植肾的存活产生重要或非常重要影响。结论通过控制影响移植肾存活的危险因素,致敏患者移植肾存活同样能取得满意效果。     

Risk factors for graft survival in sensitized recipients of kidney transplantation.

OBJECTIVE: To investigate the independent prognostic factors for graft survival in sensitized recipients undergoing kidney transplantation, so as to identify the individuals at high risk of graft loss before transplantation. METHODS: A retrospective investigation was conducted in 102 sensitized kidney transplant recipients and 31 relative variables were analyzed with SPSS10.0 software. Using log-rank method, the influence of these variables on short- and long-term graft survivals was evaluated, and Kaplan-Meier analysis was performed to estimate the 1-, 3- and 5-year graft survival rates and half-life. Proportional hazards regression analysis (Cox model) was used to assess the relative risks of the potential variables. RESULTS: In the recipients with a mean half-life of 8.9 years, the 1-, 3- and 5-year graft survival rates were 90%, 85%, and 75%, respectively. By log-rank analysis, the factors affecting short- and long-term graft survivals were identified, namely the recipient age, times of transplantation, levels of panel reactive antibody and the post-operative anti-HLA-IgG antibody, HLA mismatch, renal function, time needing for graft function recovery, presence of acute rejection, delay of graft function recovery and infection, which affected the graft survival demonstrated by Cox model multivariate analysis. CONCLUSION: High-quality donor kidney and minimization of the risk factors for graft survival may insure successful kidney transplantation in sensitized recipients.     

异基因造血干细胞移植中嵌合体的STR-PCR定量检测

目的:研究异基因造血干细胞移植后供者单个核细胞、CD3+T细胞、CD15+粒细胞植入情况。方法:采集22例清髓性外周血干细胞移植患者移植前及移植后不同时间的骨髓标本,应用免疫磁珠进行CD3+T细胞、CD15+粒细胞分选,常规提取DNA,PCR扩增后,进行毛细管电泳,确定基因位点及峰面积,计算供者嵌合率。结果:22例患者均在中位时间+12d(+9d～+15d)造血恢复。骨髓单个核细胞在+7d时供者嵌合率为70%,+14d供者嵌合率均超过90%,+21d和+28d供者嵌合率均超过95%以上。CD15+粒细胞在+7d时嵌合率为40%,在+14d、+21d和+28d供者嵌合率>95%。CD3+T细胞在+7d、+14d、+21d和+28d供者嵌合率分别为65%、80%、84%和90.9%。移植后早期完全嵌合组急性GVHD发生率高于混合嵌合组。结论:在+28d3群细胞均可达到供者完全嵌合状态。定量检测对判断早期植入、预测复发,指导临床干预治疗具有重要意义。     

移植肾急性排斥反应中外周血CD40、CD40L分子的表达及临床意义

目的 研究移植肾出现急性排斥反应时,受者外周血中白细胞CD40、CD40L分子的表达及临床意义.方法 将患者分成2组,急性排斥反应组15例,未出现排斥反应组(对照组)41例,采用流式细胞仪检测56例肾移植受者外周血CD40、CD40L在CD4 、CD8 T细胞表面的表达.结果 当发生急性排斥反应时,受者的外周血CD40、 L分子的表达均增强,排斥组与未排斥组间差异有显著性.结论 肾移植急性排斥反应的发生与CD40、CD40L共刺激通路的活化有十分密切关系,CD40、CD40L分子的表达随急性排斥反应的增加而显著上升.可作为临床上对急性排斥反应的无创性诊断方法之一.