慢性非细菌性前列腺炎/慢性骨盆疼痛综合征患者外周血T调节细胞检测及与临床症状的相关性分析

目的 探讨慢性非细菌性前列腺炎/慢性骨盆疼痛综合征(CAP/CPPS)患者外周血CD4+ CDhigh25调节性T细胞数量和功能的变化及其临床意义.方法 采用流式细胞术检测45例CAP/CPPS患者和18例健康对照者外周血CD4+ CDhigh25T细胞、CD4+ CDhigh25T细胞分别占CD4+ T淋巴细胞的百分比,ELISA法检测血清和精浆IL-6、IL-10、TGF-β1、TNF-α水平,Spearman法分析各检测指标与临床症状的相关性.结果 CAP/CPPS患者外周血CD4+ CD+25和CD4+ CDhigh25T细胞占CD:T淋巴细胞的百分比[(16.94±9.07)%,(5.98±0.57)%]与正常对照组[(15.98±3.76)%,(6.93±0.49)%]比较差异无统计学意义(P>0.05);与正常对照组比较,CAP/CPPS患者血清中TNF-α水平增加[(0.34±0.01)ng/ml与(0.11±0.01)ng/ml,P<0.053,TGF-β1水平降低[(15.17±6.35)ng/ml与(27.01±13.29)ng/ml,P<0.05],精浆中IL-6、TGF-β1及TNF-α水平增高[(0.80±0.01)ng/ml与(0.44±0.11)ng/ml,(195.07±20.46)ng/ml与(126.46±25.99)ng/ml,(0.30±0.02)ng/ml与(0.12±0.02)ng/ml,P<0.05];血清和精浆IL-6水平与美国国立卫生研究院慢性前列腺炎症状评分指数(NIH-CPSI)呈正相关(r=0.456,P<0.05;r=0.506,P<0.01),精浆IL-10水平与疼痛指数呈正相关(r=0.276,P<0.05);CD4+ CDhigh25 T细胞数与血清TGF-～1水平呈正相关(r-0.486,P<0.01),与患者年龄、病程及NIH-CPSI指数未见明显相关性(r=-0.075,P>0.05;r-=0.506,P>0.05;r=-0.112,P>0.05).结论 外周血CD4+ CDhigh25 T调节细胞数量与CAP/CPPS的发病无明显关联,其功能缺陷可能导致CAP/CPPS发病,细胞因子在CAP/CPPS的发病过程中起重要作用,并且与临床症状密切相关.     

不同分期前列腺癌患者外周血CD4~+ CD25~+ Foxp3~+调节性T细胞的变化及与胰岛素抵抗的相关性

目的 :观察不同分期前列腺癌患者外周血单个核细胞CD4+CD25+Foxp3+调节性T细胞的变化及与胰岛素抵抗的关系。方法:采用流式细胞术检测62例前列腺癌患者(患者组,临床TNM分期Ⅰ期5例、Ⅱ期16例、Ⅲ期21例、Ⅳ期20例)外周血单个核细胞(PBMC)中CD4+CD25+Foxp3+调节性T细胞数目,计算CD4+CD25+Foxp3+调节性T细胞占CD4+T淋巴细胞的百分率;并检测其空腹胰岛素及空腹血糖水平,计算胰岛素抵抗指数(HOMA-IR);采用ELISA法测定外周血胰岛素样生长因子1(IGF-1)水平,分析CD4+CD25+Foxp3+调节性T细胞与胰岛素抵抗的相关性,并与42例健康体检者进行对照。结果:与健康对照组相比,前列腺癌患者HOMAIR明显升高(6.68±1.66 vs 3.68±1.42),IGF-1水平明显下降[(96.39±21.21)ng/ml vs(164.56±30.58)ng/ml],PBMC CD4+CD25+Foxp3+Treg占CD4+T淋巴细胞的百分率[(13.88±0.96)%vs(5.33±0.65)%]及CD4+CD25+Foxp3+Treg绝对值[(3.55±0.29)×107vs(1.99±0.78)×107]明显升高(P0.05,P0.01)。患者PBMC CD4+CD25+Foxp3+Treg占CD4+T淋巴细胞的百分率及CD4+CD25+Foxp3+Treg绝对数﹑HOMA-IR均随TNM分期逐渐加重而增加,IGF-1逐渐下降;相关性分析表明:CD4+CD25+Foxp3+Treg/CD4+T及CD4+CD25+Foxp3+Treg绝对数均与HOMA-IR呈明显正相关(r分别为0.689、0.722,P0.01),与IGF-1呈明显负相关(r分别为-0.896、-0.747,P0.01)。结论:前列腺癌患者存在不同程度的胰岛素抵抗,且随着疾病程度的加重,外周血CD4+CD25+Foxp3+调节性T细胞数目和比例及胰岛素抵抗逐渐加重;CD4+CD25+Foxp3+调节性T细胞可能通过调节胰岛素抵抗参与其形成和发展。     

CD4~+CD25~+调节性T细胞诱导同种异体复合组织移植免疫耐受功能的研究

目的 探讨供体抗原特异性CD4~+CD25~+调节性T细胞(Treg),对同种异体复合组织 移植(CTA)受体大鼠细胞免疫耐受功能的影响.方法 采用免疫磁珠法分选CD4~+CD25~+Treg,将数 量1×10~6的CD4~+CD25~+Treg输注入CTA受体的大鼠,术后30 d采取外周血,尼龙毛柱分离T、B细 胞,检测在IgM 抗体刺激下的增殖放射强度的液体闪烁测定(CPM)值及血清lgG和IgA水平,判断 CD4~+CD25~+Treg对T、B细胞功能的影响及CTA的存活情况,并进行统计学分析.结果 分选的CD4~+CD25~+Treg纯度为95.6%.T、B细胞CPM值:正常对照组分别为2436±358、2418±348,Treg 局部干预组为2273±136、2252±127,Treg全身干预组分别为2338±228、2315±218,Treg未干预组分 别为3749±245、3720±224;Treg 未干预组与其三组比较差异均有统计学意义(P<0.01).血清IsG、IgA水平:Treg 局部和全身干预组分别为(12.56±1.30)g/L、(2.38±0.21)g/L和(13.48±1.23) g/L、(2.86±0.24)g/L,与正常对照组(12.35±1.28)g/L、(2.36±0.12)g/L比较差异无统计学意 义(P>0.05),三组与Treg未干预组(16.58±1.12)g/L、(3.75±0.37)g/L比较差异有统计学意义 (P<0.01).Treg 局部和全身干预组CTA存活时间分别为(97±13)和(63±lO)d,显著长于Treg未 干预组的(22±8)d(P<0.01).结论 供体抗原特异性CD4~+CD25~+Treg对T、B细胞功能有明显的 抑制作用,能诱导CTA免疫耐受,延长其存活时间,且局部应用效果优于全身.     

胃癌患者外周血调节性T细胞检测及初步分析

目的 检测胃癌患者外周血调节性T细胞(Treg)水平并对其进行初步分析.方法 四色流式细胞仪检测术前胃癌患者(n=25)及正常健康者(n=25)外周血CD4+ CD25+ FOXP3+Treg水平.结果 正常对照组CD4+ CD25+ FOXP3+ Treg/PBL为(1.229±0.656)%,胃癌组CD4+ CD25+ FOXP3+ Tres/PBL为(1.993±0.830)%.胃癌患者外周血Treg较正常对照组明显升高(P<0.01).结论 胃癌患者周血Treg水平升高,可因其免疫负调作用而导致免疫抑制.     

门静脉高压症脾亢患者外周血CD4+CD25+CD127low/-调节性T细胞含量与免疫功能研究

目的 研究门静脉高压症(PHT)脾亢患者外周血CD4+CD25+CD127low/-调节性T细胞(Treg)与CD3+、CD4+、CD8+T细胞的表达变化.探讨脾脏免疫功能改变及其可能的调节机制.方法 PHT脾亢患者20例(脾亢组),健康成人18例(对照组),采用流式细胞术检测外周血中CD4+CD25+CD127low/-Treg及CD3+、CD4+、CD8+T细胞的表达变化.结果 脾亢组外周血中CD4+CD25+CD127low/-Treg占CD4+T细胞的比例为(5.3±3.0)%,明显高于对照组的(2.5±0.9)%(P<0.01).脾亢组CD3+、CD4+、CD8+T细胞含量均低于对照组(P<0.05).CD4+CD25+CD127low/-Treg的含量与CD3+T细胞比例呈负相关(r=-0.630,P<0.01),与CD4+T细胞比例呈负相关(r=-0.561,P<0.05).结论 PHT脾功能亢进患者CD4+CD25+CD127low/-调节性T细胞表达量增多,可能对脾脏免疫功能的调节有重要作用.     

胃癌患者手术前后CD4+CD25+调节性T细胞的变化

目的 研究胃癌患者手术前、后调节性T细胞(Treg)及FoxP3表达的变化.方法 采用流式细胞术检测20例胃癌患者术前及其中15例接受了手术者术后1周(简称术后)以及15例因胃部不适接受胃镜检查的自愿者(正常对照组)外周血中Treg数量的变化,用RT-PCR法检测Treg的特异性分子标志物FoxP3的转录水平,同时用免疫组织化学法检测胃癌组织中FoxP3蛋白的表达情况.结果 胃癌患者术前外周血中CD4+T细胞中的CD4+ CD25+比例明显高于正常对照组[(19.39±5.58)%比(9.91±3.23)%,P＜0.01],而术后CD4+ CD25+比例较术前明显下降[(13.50±5.93)%比(19.39±5.58)%,P＜0.05].胃癌患者术前外周血中FoxP3转录水平明显高于正常对照组(0.86±0.03比0.64±0.02,P＜0.01),而术后较术前明显下降(0.73±0.04比0.86±0.03,P＜0.05),提示FoxP3转录水平与Treg变化一致.胃癌患者外周血中CD4+T细胞在单个核细胞中的比例与正常对照组相比明显下降(P＜0.01),而手术前、后变化不明显.20例胃癌患者中13例胃癌癌细胞的细胞浆中有不同程度的FoxP3蛋白表达(强阳性2例,中阳性6例,弱阳性5例),7例胃癌患者的胃癌细胞中不表达.结论 Treg可能通过免疫抑制作用在胃癌的发生、发展中发挥作用,肿瘤组织本身可能是引起Treg变化的重要始动因素.     

肝癌病人外周血中CD4+CD25+FOXP3+调节性T淋巴细胞表达水平的临床研究

目的 了解CD4+CD25+FOXP3+T调节性T细胞在肝癌病人外周血中的表达水平并探讨其临床意义.方法 应用流式细胞术测定18例肝癌病人外周血CD4+CD25+FOXP3+调节性T细胞占CD4+T淋巴细胞百分比,并与26例l临床对照者和24例健康对照者进行比较.结果 肝癌病人外周血中CD4+CD25+T细胞占CD4+T细胞百分比(4.25%±3.98%)明显高于临床对照组(1.34%±1.14%)及健康对照组(1.29%±0.95%)(P＜0.01),而两个对照组之间并无显著性差异(P＞0.05).CD4+CD25+FOXP3+T细胞在肝癌病人外周血所占CD4+T细胞比率(2.94%±0.91%)也较临床对照组(0.76%±0.34%)及健康对照组(0.81%±0.29%)显著升高(P＜0.001),且升高幅度强于CD4+CD25+T细胞水平,两个对照组之间并无显著性差异(P＞0.05).结论 CD4+CD25+FOXP3+T细胞是更为准确的调节性T细胞,其在肝癌病人外周血中表达水平明显升高,检测CD4+CD25+FOXP3+T水平对肝癌的预防治疗具有重要临床意义.     

小鼠CD4+CD25+T调节细胞的分离、纯化及其功能检测

目的 探讨小鼠CD4+CD25+T调节细胞(Treg)的分离培养、纯化及其部分功能检测.方法 采用免疫磁珠分离法(MACS)对分离小鼠的脾淋巴细胞进行分选CD4+CD25+Treg细胞,锥虫蓝细胞染色检测其活性,流式细胞仪检测分选所得活性细胞的纯度,酶联免疫吸附试验(ELISA)检测培养上清液中白细胞介素(IL)-2、IL-10水平的浓度.结果 MACS分离的CD4+CD25+Treg细胞的纯度达83%～96%.体外培养中Treg组、T组和混合组IL-2和IL-10的平均水平分别为:(10.25±2.31)、(40.32±8.05)ng/L;(5 8.21±13.05)、(11.52±3.01)ng/L;(39.54±12.82)、(31.25±4.36)ng/L,数据差异有统计学意义(P＜0.05,P＜0.01).结论 采用MACS系统两步法,可获得高纯度、具有免疫抑制功能的Treg细胞,该细胞对CD4+CD25-T细胞的免疫抑制作用可能是通过IL-10对IL-2的调节作用实现的.     

CD4+CD25+FOXP3+T细胞在肝癌肝移植肿瘤复发中的作用

目的 探讨肝癌肝移植病人移植前后外周血和肿瘤组织中CD4+CD25+FOXP3+T细胞比例变化及其临床意义.方法 用流式细胞仪检测肝癌肝移植病人和其他肝移植病人术后外周血中CD4+CD25+FOXP3+T细胞的比例,并采用正常人作对照.用免疫组化法检测肝癌病人和非肝癌病人肿瘤组织中FoxP3的表达及CD8+T细胞浸润的比例.观察肝癌肝移植病人术后及肿瘤复发后调节性T细胞的变化及其对肿瘤复发的影响.结果 流式细胞检测显示肝癌肝移植、非肝癌肝移植的病人术后外周血中CD4+CD25+FOXP3+T细胞占CD4+T细胞的比例较正常人明显升高,分别为(10.15±1.00)%、(5.30±1.64)%和(3.20±1.18)%,P<0.05.肝癌肝移植肿瘤复发病人较未复发病人外周血CD4+CD25+FOXP3+T比例明显升高,分别为(15.15±1.50)%和(6.80±1.50)%,P<0.01.免疫组化检测显示肿瘤组织中FOXP3+T细胞增多,CD8+T细胞浸润明显减少.结论 肝癌肝移植肿瘤复发的病人外周血中CD4+CD25+FOXP3+T细胞比例升高.调节性T细胞可能通过减少CD8+T细胞浸润,加速肿瘤复发.     

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.     

Evaluation of lymphocytes CD4+ and CD8+ and expression of ZAP-70 kinase on CD3+ and CD19+ lymphocytes in obese patients undergoing laparoscopic cholecystectomy

Background

Obesity has become a global epidemic and a leading metabolic disease in the world. Laparoscopic surgeries may influence the function of the immunologic system. The percentages of CD4+ and CD8+ T lymphocyte cells have been described as prognostic factors for patients undergoing abdominal surgeries. This study aimed to evaluate the changes in CD4+ and CD8+ T lymphocyte cells, the ratio of CD4+ to CD8+ cells, and the ZAP-70 kinase expression on T CD3+ and B CD19+ cells in obese and normal-weight individuals undergoing laparoscopic cholecystectomy (LC).

Methods

The study group consisted of 46 asymptomatic patients with gallstones shown by ultrasound examination but without signs of any gallbladder complications. The patients underwent planned LC. Blood samples were obtained at three times, and the percentages of studied cells were measured by flow cytometry. Patients were enrolled to two groups: N group (body mass index [BMI], ≤25 kg/m²) and O group (BMI, ≥30 kg/m²). For statistical analysis, the Mann–Whitney U test and the Wilcoxon matched-pairs signed-ranks test were used. All p values lower than 0.05 were considered significant.

Results

The percentage of CD4+ T cells did not differ between the N and O groups before or after the surgery. Only in the N group did the percentage of CD4+ lymphocytes increase from 0 to 48 h. A higher percentage of CD8+ lymphocytes was observed in the O group postoperatively than in the N group. Differences of ZAP-70 kinase expression in the O group were observed at 24 and 48 h of the study. Decreased expression of ZAP-70 kinase was shown in the N group at both 0–24 and 24–48 h. In the O group, this tendency was noted at 24–48 h.

Conclusions

Immunologic activation after LC was confirmed in both weight groups. However, higher modulation, more typical for open surgeries, was observed in the obese group.     

肝癌、肝硬化组织中CD3、CD57、CD20细胞数量与临床病理表现及预后的关系

目的探讨CD3、CD57、CD20细胞在原发性肝细胞癌(HCC)、癌旁、肝硬化及正常肝组织中的数量及意义.方法HCC 60例,单纯性肝硬化62例,正常肝组织23例,以免疫组化SP法进行CD3、CD57、CD20染色,对阳性细胞数进行定量分析并与临床资料进行相关探讨.结果(1)各组CD3+细胞平均数从高到低为癌旁组织、癌组织、肝硬化组织、正常肝组织(P＜0.05);各组CD57+细胞平均数从高到低为癌组织、癌旁组织、正常肝组织、肝硬化组织(P ＜0.05);各组CD20+细胞平均数从高到低为癌组织、癌旁组织、肝硬化组织、正常肝组织(P ＜0.01).(2)HCC中CD3+细胞、CD57+细胞、CD20+细胞与组织学分级均无明显关系.(3)HCC中CD57+细胞和CD20+细胞随着临床分期的发展有下降的趋势(P ＜0.05);HCC中CD3+细胞平均数与临床TNM分期无关.(4)HCC中15月内有转移组的CD57+、CD3+细胞数均少于无转移组(P＜0.01).HCC患者15月内有无转移与HCC和癌旁组织中的B细胞分布均无关.结论临床上,随着HCC患者的病情恶化,CD3+、CD57+、CD20+细胞逐渐减少.CD3+、CD57+、CD20+细胞可成为反映机体抗肿瘤特异性细胞免疫状态和生物学行为及判断患者预后的重要指标.     

Acceleration of apoptosis in CD4+CD8+ thymocytes by rapamycin accompanied by increased CD4+CD25+ T cells in the periphery

BACKGROUND: Rapamycin (Rapa) is an immunosuppressant that is used in patients and animal models to control allograft rejection. Its mechanisms of action are not fully understood. In this article, the authors have investigated the effects of therapeutic doses of Rapa on both thymic and peripheral T-cell populations in the adult rat. METHODS: The therapeutic dosage of Rapa was optimized using cardiac transplantation between LEW and DA rats. Thymic morphology was assessed by hematoxylin-eosin staining. Flow cytometric analysis was performed to analyze T-cell phenotype and apoptosis. T-cell receptor (TCR)-mediated T-cell responsiveness was evaluated by 3[H]-thymidine deoxyribose incorporation. RESULTS: Rapa induced atrophy in the thymus but not in peripheral lymphoid organs. Moreover, fibrosis occurred in thymus that was long-lasting after Rapa withdrawal. In animals treated with Rapa, there was a significant reduction in CD4+CD8+ thymocytes caused by accelerated apoptosis, whereas CD4-CD8-, CD4+CD8-, and CD8+CD4- populations remained unaffected. In contrast, the cellularity of the periphery lymphoid organs was not altered. Within the CD4+ thymocyte population, CD4+CD25+ thymocytes were resistant to Rapa-accelerated apoptosis, and in the periphery, the ratio of CD4+CD25+ to CD4+CD25- T cells was increased. Notably, the peripheral CD4+CD25+ T cells were hyporesponsive to TCR-mediated activation. CONCLUSIONS: The resistance of the peripheral CD4+CD25+ T cells to Rapa treatment might contribute to its immunosuppressive action. The long-term effects of Rapa on thymus atrophy and thymocyte development requires consideration with respect to its clinical application.     

Effect of hormone replacement therapy on CD4+ and CD8+ numbers, CD4+/CD8+ ratio, and immunoglobulin levels in hemodialysis patients

Uremia induces a suppression of the immune status. A large clinical literature suggests that estradiol (E2) plays a critical role in immune function. A large proportion of women hemodialysis patients faced early menopause and inadequate estrogen levels. The aim of the present study is to evaluate the effect of hormone replacement therapy on immune function in terms of CD4+ numbers (inducer/helper T cells), CD8+ numbers (cytotoxic/suppressor T cells), CD4+ / CD8+ ratio, and IgG, IgM, IgA levels in woman hemodialysis patients. In our study, 15 female hemodialysis patients (median age 32.6 range 24-45) were treated with triphasic estrogen/progesterone preparation (estradiol 2 mg for 10 days, and afterwards estradiol 2 mg+norethisterone 1 mg for another 10 days, and at the end estradiol 1 mg for 6 days) for 6 months. CD4+ numbers, CD8+ numbers, and IgG, IgA, and IgM levels were determined before and after HRT. The "paired-samples T" test was used for statistical analysis of pretreatment and posttreatment values. A significant increase was observed for CD4+ numbers (582 +/- 435 versus 637 +/- 445, p = 0.04) and CD4+/CD8+ ratio (1.4 +/- 0.16 to 2.4 +/- 0.3, p < 0.01) after hormone replacement therapy (HRT). Serum immunoglobulin levels were not changed significantly. In conclusion, in postmenopausal hemodialysis patients, HRT significantly increased CD4+ numbers and CD4+ / CD8+ ratio, but no effect was observed in IgM, IgG, and IgA levels. Long-term clinical effects of HRT on immune system should be investigated in dialysis patients with further studies.     

大鼠CD4^＋CD25^＋调节性T细胞的分离及功能鉴定

目的：研究利用免疫磁珠分选法稳定分离正常大鼠脾脏CD4^＋CD25^＋调节性T细胞的方法。方法：采用免疫磁珠两步法分离大鼠脾组织CD4^＋CD25^＋T细胞。首先采用藻红蛋白（PE）标记的抗CD25抗体和抗PE多功能磁珠试剂盒阳性分选CD25^＋T细胞,再用抗异硫氰酸荧光素（FITC）标记抗体和抗IgG磁珠阳性分选获得CD4^＋CD25^＋T细胞。分离后的细胞经流式细胞仪检测分离纯度,台盼蓝染色检测细胞存活率,体外增殖实验检测其对CD4^＋CD25^-T细胞的免疫抑制作用。结果：两次阳性分选后获得的CD4^＋CD25^＋T细胞纯度为（90.4±1.6）%,细胞存活率为（92.6±2.4）%。体外增殖实验表明,CD4^＋CD25^＋T细胞能明显抑制CD4^＋CD25^-T细胞的增殖（P〈0.01）。结论：采用免疫磁珠法两次阳性分选,可稳定地获得纯度理想并有免疫抑制功能的大鼠CD4^＋CD25^＋T细胞。     

Differentiation of human alloreactive CD4+ and CD8+ T cells in vitro

BACKGROUND: Recent studies have revealed that, during viral infection, ordered phenotypic and functional changes occur in human antigen-specific T cells. We analyzed whether a similar differentiation program is induced after alloantigen stimulation in vitro. METHODS: Peripheral blood mononuclear cells and purified CD4(+)CD45RA+, CD4(+)CD45RO+, and CD8+ T cells from healthy individuals were labeled with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE). Cells were co-cultured with allogeneic irradiated cells. Flow cytometric analysis was performed on days 3, 5, and 7 of culture using surface CD45RA, CD27, CD28, CCR7, and intracellular perforin and granzyme B markers in relation to CFSE dilution. RESULTS: Based on the decrease in CFSE fluorescence, both CD4+ and CD8+ T cells showed an early and vigorous response to allogeneic stimulation. Loss of CD45RA expression and up-regulation of CD27 and CD28 costimulatory molecules was an early event occurring in the first generations of dividing cells. Differentiation at later stages of proliferation was characterized by loss of CCR7 homing receptor expression, more pronounced in CD4+ than in CD8+ T cells, indicating the decreased ability of these cells to traffic to secondary lymphoid organs. Production of the cytotoxic effector molecules perforin and granzyme B increased with the number of cell divisions. CONCLUSIONS: Our data thus show that short-term phenotypic and functional changes of alloreactive T cells follow the differentiation pattern seen in the early stages of an antiviral immune response.     

The role of CD8+ and CD4+ cells in islet allograft rejection

The requirements of CD8+ and CD4+ cells for islet graft rejection in combinations with different histoincompatibilities were investigated by in vivo administration of anti-Lyt-2.2 (CD8) mAb, anti-L3T4 (CD4) mAb, or both to recipient mice. In B10.AQR----B10.A (H-2K-incompatible) and B10.A(5R)----B10.A (H-2K- and IA-incompatible) combinations, administration of either anti-Lyt-2.2 (CD8) or anti-L3T4 (CD4) mAb completely blocked islet graft rejection, indicating that neither CD8+ cells nor CD4+ cells alone were capable of mediating rejection, and that collaboration of CD8+ cells and CD4+ cells was necessary. On the other hand, in the BALB/c----B6 (H-2- and non-H-2-incompatible) combination, administration of anti-Lyt-2.2 (CD8) or anti-L3T4 (CD4) mAb resulted in rejection of most of the grafts, although survival was prolonged significantly, and administration of both anti-Lyt-2.2 (CD8) and anti-L3T4 (CD4) mAb together completely blocked rejection. These results suggested that either CD8+ or CD4+ cells were capable of mediating rejection, but that rejection was maximal in the presence of both T cell subsets. Immunohistochemical analyses showed marked depletion of CD8+ cells and CD4+ cells in grafted islets as well as spleens when anti-Lyt-2.2 (CD8) and anti-L3T4 (CD4) mAb, respectively, were injected.