CD4+T细胞ATP含量与肝移植后急性排斥反应的关系

目的 探讨CD4+T细胞ATP含量与肝移植术后围手术期急性排斥反应(acute rejection,AR)的关系.方法 以2009年2月至2009年10月在本院行肝移植术的77例病人为研究对象.肝移植病人术前以及术后1、2、4周各采集1份全血标本,发生AR当日和激素冲击治疗后1周各采集1份全血标本.每次均送检1～2份健康志愿者全血标本作为对照.用ImmuKnowTM免疫细胞功能测定试剂盒检测样本中CD4+T细胞ATP值.结果 AR组病人ATP值在术后第1周达到高峰,显著高于同期非排斥组.术后第1周的高ATP值对诊断肝移植术后围手术期AR具有较好的敏感度和特异度.AR组病人排斥当日的ATP值与排斥活动指数(RAI)呈显著正相关,经激素冲击治疗1周后,AR均达到逆转,ATP值明显降低.结论 肝移植术后围手术期,CD4+T细胞ATP值动态变化与移植肝急性排斥反应密切相关,有望成为诊断和预防围手术期AR发生以及抗排斥治疗效果的无创监测指标.     

供体肝脏转染CD40抑制大鼠肝移植急性排斥反应的研究

在器官移植排斥反应中 ,细胞免疫起重要的作用。T细胞激活必须接受抗原呈递细胞 (antigenpresentingcell,APC)的双重信号激活 ,其中第二信号为T细胞抗原特异性激活所必需 ,若缺乏共同刺激信号 ,可导致T细胞的免疫无应答甚至凋亡(apoptosis) [1] 。本实验研究利用供体肝脏转染pCDNACD4 0Ig ,供体肝脏表达CD4 0Ig ,阻断共同刺激通路 ,观察CD4 0Ig对同种异体肝移植排斥反应的抑制作用。材料和方法1 .实验材料 :TUNEL试剂盒购于德国宝灵曼公司 ,鼠抗人CD4 0单克隆抗体购于Zymed公司 ,PcDNA 3.1CD4 0Ig为人CD4 0胞外区与人IgGFC…     

调节性CD4+T细胞在大鼠自发肝移植耐受中的作用

目的 探讨在肝移植的自发耐受模型中，调节性CD4^＋T细胞的免疫抑制作用机制。方法 利用近交系大鼠从Lewis（LEW）到Wistar Furth（WF）的肝移植组合，对移植后不同时期的宿主注射抗CD4的单克隆抗体（Anti-CD4mAb），然后抽血检测丙氨酸氨基转氨酶（ALT）的动态变化；并结合细胞毒性T淋巴细胞（CTL）试验了解宿主脾细胞中T细胞亚群的动态改变。结果 对肝移植自然生存的宿主注射Afiti—CD4mAb后，术后第21天、42天均能够诱导出肝损害（排斥反应），但第56天、100天以上的则未能诱导出来，且该损害能被抗CD8单克隆抗体阻断。另外CTL试验显示宿主的脾细胞中，初始型CTL前体细胞在移植56d后未能检测出来。结论 在自发性肝耐受模型中。宿主术后早期存在由CD4^＋T细胞介导的下调原始效应性T细胞的作用机制。     

肝移植急性排斥反应者外周血CD4+CD25+Foxp3+调节性T淋巴细胞的变化

目的 探讨良性终末期肝病患者肝移植术后外周血CD4+CD25+叉状头螺旋转录因子(Foxp3)+调节性T淋巴细胞在急性排斥反应期的变化及意义.方法 2004年12月至2008年1月间,符合入选条件的良性终末期肝病患者共55例,按照术后是否发生急性排斥反应分为排斥组(14例)和无排斥组(41例).肝移植术前用流式细胞仪检测患者外周血CD4+CD25+Foxp3+T淋巴细胞占CD4+T淋巴细胞的百分率(简称CD4+CD25+Foxp3+T细胞百分率),出院后1年内每隔3～6个月复查;发生急性排斥反应时,于治疗前和治疗缓解后(3～6个月)复查.比较两组患者外周血CD4+CD25+Foxp3+T细胞百分率的变化,对排斥组发生急性排斥反应时外周血CD4+CD25+Foxr3+T细胞百分率与排斥反应活动指数(RAI的相关性进行统计学分析.结果 肝移植术前,排斥组与无排斥组外周血CD4+CD25+Foxp3+T细胞百分率的差异无统计学意义(P＞0.05).排斥组患者发生急性排斥反应时外周血CD4+CD25+Foxp3+T细胞百分率为(2.23±0.54)%,低于无排斥组的(2.99±0.86)%,差异有统计学意义(P＜0.01).排斥组中,患者发生急性排斥反应时外周血CD4+CD25+Foxp3+T细胞百分率低于未发生急性排斥反应时的(3.67±0.70)%,差异有统计学意义(P＜0.01).排斥组患者发生急性排斥反应时外周血CD4+CD25+Foxp3+T细胞百分率与RAI呈负相关(r=-0.80,P＜0.01).结论 监测肝移植受者外周血CD4+CD25+Foxp3+调节性T淋巴细胞的变化,可辅助诊断急性排斥反应及判断其严重程度.

Abstract：

Objective To investigate the expression of peripheral blood (PB) CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) in patients with benign end-stage liver disease after liver transplantation and the relationship between levels of PB Tregs and acute rejection. Methods A prospective analysis was performed on 55 consecutive patients who underwent liver transplantation.Fourteen out of 55 cases suffered from acute rejection after liver transplantation were defined as rejection group,while the rest patients were classified into no acute rejection group. PB was obtained from liver transplant patients at different time points longitudinally: pre-transplant, post-transplant within one year and acute rejection. The circulating CD4+ CD25+ Foxp3+ Tregs in PB were measured by flow cytometry. Blood samples were drawn during acute rejection, at the same time, liver biopsies were performed. The circulating CD4+ CD25+ Foxp3+ Tregs were compared between two groups.Results There was no difference between two groups in levels of circulating CD4+ CD25+ Foxp3 + Tregs cells pre-transplant. However, the levels of circulating CD4+ CD25+ Foxp3+ Tregs in rejection group were decreased significantly as compared with no-rejection group (2. 23 % ± 0. 54 % vs. 2. 99 % ±0. 86 %,P＜0.01). The frequency of CD4+ CD25+ Foxp3+ T cells was negatively correlated with rejection activity index (RAI) (r = - 0. 80, P＜0. 01 ). Conclusion Monitoring PB CD4+ CD25+ Foxp3+ Tregs levels may be helpful in evaluating the immune state and act as a more sensitive marker for acute rejection diagnosis in the patients following liver transplantation.     

CD4+CD25+调节性T细胞在肝移植术后腹腔感染中的表达及其作用

目的研究移植后细菌感染CD4^＋ CD25^＋调节性T细胞（Treg）表达的变化。方法以DA大鼠为供体,LEW大鼠为受体,采用改良的Kamada二袖套法建立肝移植模型。术后随机分为3组,G1组术后3d腹腔生理盐水注射;G2组术后3d给予腹腔大肠杆菌注射;G3组术后常规给予免疫抑制药物CsA,3mg/（kg·d）。感染后7d处死动物,每组6个样本,检测血清ALT、TB,并采用流式细胞术检测脾脏Treg的水平,RT-PCR方法检测脾脏Foxp3和肝脏IL-10、TGF-β mRNA的表达。结果G3、G2组Treg表达水平显著升高,分别为：（16.72±0.95）%,（27.4±1.09）%,与G1（9.88±0.98）%比较差异均有统计学意义（P〈0.01）;脾脏Foxp3和肝脏IL-10、TGF-β mRNA表达水平均增加;感染组的肝功虽然较差,与排斥组相比无统计学意义,但从病理切片观察该组肝脏的急性免疫排斥反应减轻,Banff评分有统计学意义。结论大鼠肝移植术后腹腔细菌感染增加了脾脏Treg的表达,促进了免疫抑制作用的发挥,部分减轻了急性排斥反应。     

T细胞疫苗抗异品系大鼠肝移植排斥反应的实验研究

目的 研究特异性T细胞疫苗接种对大鼠肝移植后排斥反应的影响。方法 利用LOU/CN大鼠的脾细胞免疫CHN大鼠,取后者脾细胞活化制备T细胞疫苗,对肝移植受者CHN大鼠进行免疫。测定T细胞疫苗接种与未接种组的混合淋巴细胞培养cpm值。采用三袖套法进行大鼠原位肝移植。按移植前后处理方法不同分为3组:（1）T细胞疫苗接种组:用T细胞疫苗接种后7～9d的CHN大鼠进行原位肝移植;（2）环孢素A（CsA）组:未接种T细胞疫苗的CHN大鼠进行原位肝移植后,用CsA10mg·kg-1·d-1进行免疫抑制治疗;（3）阴性对照组:未接种T细胞疫苗的CHN大鼠进行原位肝移植后,用生理盐水作对照。分别测定各组的存活时间、微量细胞毒反应。结果T细胞疫苗接种组与未接种组外周血淋巴细胞cpm值分别为:9804±1182和22386±2117,二者比较,差异有显著性（P<0.01）。异品系大鼠肝移植后,T细胞疫苗接种组存活时间为（17.1±2.8）d,CsA组为（14.2±3.1）d,阴性对照组为（11.6±2.4）d。三组比较,（P<0.01）。微量细胞毒测定死细胞百分率:T细胞疫苗接种组为（26.4±9.3）％,CsA组为（43.7±10.4）％,阴性对照组为（62.4±8.1 ％。三组比较,差异有显著性（P<0.01）。结论T细胞疫苗接种能显著延长异品系大鼠肝移植的存活期,有显著的抗排斥反应作用。     

输注供者CD8+CD28-调节性T淋巴细胞抑制大鼠肝移植急性排斥反应的作用

目的 评价具有免疫抑制作用的CD8+CD28-调节性T淋巴细胞(Treg)体内输注在抑制大鼠肝移植急性排斥反应中的作用.方法 建立近交系大鼠肝移植自发耐受及急性排斥反应模型.从肝移植自发耐受模型受者脾脏中分离CD8+CD28-Treg,于急性排斥反应模型建立前1 d输注给受者,比较不同的输注组间受者的存活时间和移植肝病理学表现.结果 来自自发耐受模型(LEW大鼠为供者,DA大鼠为受者)的CD8+CD28-Treg输注可以延长急性排斥反应模型(LEW大鼠为供者,BN大鼠为受者)受者的存活时间,由(14.0±2.2)d延长至(24.0±3.0)d(P＜0.01),移植肝病理学显示排斥反应程度减轻.结论 大鼠肝移植自发耐受模型受者体内诱导的CD8+CD28-Treg具有抑制急性排斥反应的作用,该免疫抑制作用具有抗原特异性.     

大鼠肝移植急性排斥反应时T淋巴细胞免疫相关基因表达的基因芯片研究

目的 从基因水平了解T淋巴细胞在急性排斥反应中作用的分子机制。方法 建立大鼠肝移植急性排斥反应模型，并设对照组，利用4096条大鼠cDNA克隆的基因芯片从来自排斥组和对照组的T淋巴细胞中抽提、纯化mRNA，逆转录成cDNA，荧光标记后与芯片杂交，扫描后筛选出差异表达的基因。结果 在4096个基因中共发现差异表达基因190条，其中10条差异表达明显、已知功能的免疫相关基因为：主要组织相容性复合物(3条)和CD3抗原(1条)相关基因；干扰素调节因子1、上皮生长因子受体、转化生长因子相关基因各1条；白细胞蛋白酶抑制因子、急性期蛋白抑制因子3相关基因各1条；补体因子1基因1条。结论 T淋巴细胞在肝移植术后急性排斥反应中的作用机制涉及多个基因；基因芯片技术是一种高效、稳定的方法。     

细胞毒T淋巴细胞在异种肝移植急性排斥反应中的作用

目的：探讨异种肝移植中细胞毒T淋巴细胞（CTL）参与急性排斥反应的机制。方法：三袖套法建立仓鼠到大鼠原位肝移植模型；免疫组化方法检测同基因组与异种移植组（XT组）移植肝术后CD8和CD4型淋巴细胞浸润及Fas-L，穿孔素的表达情况，并运用原位末端标记法检测移植肝细胞的凋亡情况。结果：XT组移植肝术后第2d汇管区出现淋巴细胞浸润，逐日增多，至术后5－6d达高峰；术后第4d穿孔素及Fas-L开始表达，术后第5－6d达高峰；穿孔素的表达始终高于Fas-L。结论：异种肝移植中T淋巴细胞参与了其免疫排斥反应，通过表达穿孔素或Fas-L使靶细胞坏死或凋亡。     

大黄素对大鼠肝移植后CD4~+CD25~+调节性T细胞的影响

目的研究大黄素对大鼠肝移植后CD4+CD25+调节性T细胞的比例以及对其免疫抑制功能的影响。方法双袖套法建立大鼠原位肝移植模型,以大黄素和环孢素A(CsA)分别在术后腹腔给药,并以给予PBS作为模型对照组,观察不同药物移植后大鼠存活时间的影响,以及对移植术后大鼠外周血中CD4+CD25+调节性T细胞(Tregulatory cells,Treg)亚群的变化以及免疫功能的影响。结果大黄素能显著延长大鼠原位肝移植的术后成活时间,大黄素组大鼠的平均存活时间(17.4±2.5)d与肝移植模型对照组(8.8±1.9)d相比差异具有统计学意义;大鼠肝移植后大黄素给药可显著上调受体大鼠外周血以及肝内CD4+CD25+Treg的比例,并显著增强CD4+CD25+Treg抑制效应性T细胞的增殖能力(P0.05)。结论大黄素能够延长大鼠原位肝移植的术后成活时间,并可能通过上调外周血以及肝内CD4+CD25+Treg的数量及免疫抑制功能来实现移植后免疫排斥反应的抑制。     

Primary liver disease as a determinant for acute rejection after liver transplantation

Background: Graft rejection and infection remain major problems following liver transplantation; both are heavily influenced by the immunosuppressive regimen. Despite the disparity in the primary disease leading to transplantation, all patients receive the same post-transplant immunosuppressive treatment in a given center. The aim of this study is to detect a possible effect of the underlying disease on the incidence of early acute rejection episodes after orthotopic liver transplantation (OLT). Patients and Methods: Retrospective analysis on all 101 consecutive liver transplants performed in 95 patients between 1983 and March 1998; five of these patients, surviving less than 30 days, were not included. The immunosuppressive regimen was based on conventional triple therapy during the whole study period. The diagnosis and treatment of acute rejection within the first 30 days post-OLT was uniform throughout the whole study period. Results: Though there were no differences with respect to patients' characteristics [age, child classification, number of HLA-mismatches or cytomegalovirus (CMV)-serocompatibility], patients with primary biliary cirrhosis (PBC) showed a significant increase of acute rejection after OLT compared with the other patients transplanted for other liver diseases (P = 0.024). The incidence of infection was not elevated in patients transplanted for PBC when compared with other diagnoses. Conclusion: Our results indicate that primary liver disease may be a determinant for acute graft rejection in PBC. Furthermore, these results suggest that immunosuppressive regimens based on the underlying disease should be considered. Received: 19 October 1998 Accepted: 28 January 1999     

肝移植术后排异反应的诊断和鉴别诊断

目的 探讨肝移植术后排异反应的诊断和鉴别诊断标准。方法 对于5例肝移植术后出现并发症的病人，进行了临床表现、实验室检查以及肝脏活检病理检查的综合分析，寻找其临床规律和组织学特征。结果 （1）急、慢性排异反应在临床表现和病理所见上均有其特征性改变和诊断标准。（2）其他并发症，如纤维化胆汁淤滞性肝炎和肝动脉血栓形成，也有各自的临床病理特点。（3）肝移植术前应做供肝质量评定。结论 了解和掌握排异反应的诊断标准、鉴别诊断要点以及其他并发症的临床病理特征非常必要。     

Adjuvant treatment with ursodeoxycholic acid reduces acute rejection after liver transplantation

Acute rejection, occurring with a reported frequency of 50–70%, is still a dominating problem after liver transplantation. Medication with ursodeoxycholic acid (UDCA) has beneficial effects in different cholestatic conditions and has also been shown to reduce HLA class I antigen expression on hepatocytes in patients with PBC. Since August 1989 we have consecutively treated all patients with primary graft function with UDCA (n = 41). Patients transplanted in the first half of 1989 served as a control group (n = 8). All patients in this study were given sequential quadruple drug immunosuppression. The treatment group were given oral UDCA 10 mg/kg per day. During the first postoperative month, 17% of the UDCA-treated patients had an episode of acute rejection compared with 75% of the control patients (P < 0.01). Liver biochemistry tests 1 month postoperatively were significantly better in patients treated with UDCA. The results suggest that adjuvant treatment with UDCA reduces acute liver graft rejection.     

CD4+CD25+T细胞对同种异体小鼠心脏移植的免疫调节作用

目的 观察CD4+CD25+T细胞(Treg)对小鼠同种异体心脏移植的免疫调节作用.方法 流式细胞仪检测正常小鼠和胸腺切除+PC61小鼠淋巴结、脾脏和外周血的Treg的比例.将供体鼠BALB/C心脏移植到受体鼠B6腹腔内,观察对照组(n=6)、胸腺切除组(THY,n=8)、hCTLA4Ig组(n=8)、DST+hCTLA4Ig组(n=8)和THY+PC61+DST+hCTLA4Ig组(n=6)小鼠心脏移植后生存时间和移植心脏病理学检查.结果 正常B6小鼠淋巴结、脾脏和外周血的Treg的比例分别为5.1%、4.5%和1. 7%,明显高于胸腺切除+PC61处理组(1. 8%、1.7%、0.7%).移植心脏平均存活时间在对照组和胸腺切除组分别为(8.2±2.9)d和(7.6±3.0)d,两组间差异无统计学意义(P＞0.05);而在hCTLA4Ig组和DST+hCTLA4Ig组分别为(43.0±11.8)d和(135.0±29.7)d,均较对照组或胸腺切除组明显延长(P＜0.01);THY+PC61+DST+hCTLA4Ig组移植心脏平均存活时间(25.8±8.9)d,也明显较对照组明显延长,但短于hCTLA4Ig组和DST+hCTLA4Ig组(P＜0.01).DST+hCTLA4Ig组移植的心脏存活时间(135.0±29.7)d明显高于其他各组(P＜0.01),其病理组织学表现为间质内有较多的淋巴细胞浸润,伴毛细血管增生,管壁增厚,间质纤维化.结论 CD4+CD25+T细胞水平对同种异体心脏移植的免疫耐受具有免疫调节作用.

Abstract：

Objective To investigate the immunoregulation effects of CD4 + CD25 + T cells in mice heart allograft transplantation. Methods Flow cytometry was used to analyze the contents of CD4 + CD25 +T regulatory cells (Tregs) of the lymph nodes, spleen and blood in the normal mice group and the thymusectomy (THY) + PC61 group. BALB/C mice served as the donors and C57BL/6 (B6) mice as the recipients. Five groups were established, including control group ( n = 6 ), THY group ( n = 8 ), hCTLA4Ig group ( n = 8 ), DST ( donor-specific T-depleted spleen cells) + hCTLA4Ig group ( n = 8) and THY + PC61+ DST + hCTLA4Ig group (n = 6). The survival time after heart allograft transplantation was observed and pathological examination was done in different groups. Results In control group, the rate of Tregs in lymph nodes, spleen and blood was 5. 1%, 4. 5% and 1.7% respectively, which was significantly higher than in THY + PC61 group ( 1. 8% , 1. 7% and 0. 7% respectively). The average survival time of control and THY groups was 8. 2 ± 2.9 and 7.6 ± 3. 0 days respectively ( P ＞ 0. 05 ). The average survival time of hCTLA4Ig and DST + hCTLA4Ig groups was 43.0 ± 11.8 and 135.0 ± 29. 7 days respectively, which was significantly longer than in control group or THY group ( P ＜0. 01 ). The average survival time of THY +PC61 + DST + hCTLA4Ig group was 25.8 ± 8.9 days, which was significantly longer than in control group,but shorter than in hCTLA4Ig group or DST + hCTLA4Ig group ( P ＜ 0. 01 ). The survival time in DST +hCTLA4Ig group was 135.0 ± 29. 7 days, which was significantly longer than other groups ( P ＜ 0. 01 ).The pathological examination revealed that there were more lymphocytes infiltration and capillary vessel proliferation in the desmohemoblast in the transplanted heart of DST + hCTLA4Ig group. Conclusion CD4 +CD25 +T cells regulate the immune tolerance in the allograft transplantation.     

CD154 Blockade Abrogates Allospecific Responses and Enhances CD4+ Regulatory T‐Cells in Mouse Orthotopic Lung Transplant

Acute cellular rejection (ACR) is a common and important clinical complication following lung transplantation. While there is a clinical need for the development of novel therapies to prevent ACR, the regulation of allospecific effector T‐cells in this process remains incompletely understood. Using the MHC‐mismatched mouse orthotopic lung transplant model, we investigated the short‐term role of anti‐CD154 mAb therapy alone on allograft pathology and alloimmune T‐cell effector responses. Untreated C57BL/6 recipients of BALB/c left lung allografts had high‐grade rejection and diminished CD4⁺: CD8⁺ graft ratios, marked by predominantly CD8⁺>CD4⁺ IFN‐γ⁺ allospecific effector responses at day 10, compared to isograft controls. Anti‐CD154 mAb therapy strikingly abrogated both CD8⁺ and CD4⁺ alloeffector responses and significantly increased lung allograft CD4⁺: CD8⁺ ratios. Examination of graft CD4⁺ T‐cells revealed significantly increased frequencies of CD4⁺CD25⁺Foxp3⁺ regulatory T‐cells in the lung allografts of anti‐CD154‐treated mice and was associated with significant attenuation of ACR compared to untreated controls. Together, these data show that CD154/CD40 costimulation blockade alone is sufficient to abrogate allospecific effector T‐cell responses and significantly shifts the lung allograft toward an environment predominated by CD4⁺ T regulatory cells in association with an attenuation of ACR.     

自发免疫耐受大鼠移植肝内调节性T细胞的生物学特性

目的研究大鼠肝移植后自发免疫耐受的形成与移植肝内CD4~ CD25~ 调节性T细胞(Tr细胞)的关系。方法按供、受者不同将实验分为3组。急性排斥组:DA大鼠为供者,LEW大鼠为受者;自发耐受组:LEW大鼠为供者,DA大鼠为受者;同基因组:供、受者均为DA大鼠。各组均建立大鼠原位肝移植模型。分别在肝移植术后4、7、14、30和90 d时采用密度梯度离心法分离移植肝内淋巴细胞,免疫磁珠分离(MACS)法分选出CD4~ CD25~ Tr细胞;用流式细胞术(FCM)检测细胞纯度,同时分析CD4~ CD25~ Tr细胞比例的变化;体外细胞增殖试验研究CD4~ CD25~ Tr细胞对CD4~ CD25~-T细胞的免疫抑制作用。结果肝移植早期,急性排斥组和自发耐受组移植肝内CD4~ CD25~ Tr细胞比例均明显增加,其中急性排斥组增加更为明显;移植后4 d左右,两组CD4~ CD25~ Tr细胞比例开始下降,急性排斥组的下降幅度较大;移植后30 d,自发耐受组受者的移植肝内CD4~ CD25~ Tr细胞比例达到第2次高峰,约在移植后90 d时下降至正常生理水平。移植后7 d左右,急性排斥组受者均因发生排斥反应而死亡,而自发耐受组受者均存活。此外,CD4~ CD25~ Tr细胞能有效抑制CD4~ CD25~-T细胞的增殖。结论CD4~ CD25~ Tr细胞是一种具有特异免疫调节功能的T细胞亚群,其主动的免疫抑制功能可能是诱导大鼠肝移植自发免疫耐受的机制之一。