【作者】 杨明珍；

【导师】 吴德沛；

【作者基本信息】 苏州大学 ， 血液病学， 2007， 博士

【摘要】 【目的】建立CD4+CD25+Foxp3+调节性T细胞稳定的体外扩增体系,探讨CD4+CD25+Foxp3+调节性T细胞与单倍型相合造血干细胞移植GVHD的相关性以及对植入、GVHD、GVL、长期生存的影响;探讨HLA-Cw位点在单倍型相合造血干细胞移植中的作用,为优化供体的选择提供有价值的指标。【方法】在论文的第一部分,采用磁珠分选BalB/C小鼠脾脏中的CD4+T细胞,2ug/mlαCD3单克隆抗体包板,在含有1ug/mlαCD28单克隆抗体、rhIL-2 100U/ml和终浓度为10nM雷帕霉素RPMI-1640完全培养基中培养3周,流式细胞仪检测CD4+CD25+T细胞的表达,磁珠分选CD4+CD25+T细胞,实时定量PCR检测Foxp3 mRNA的表达,单向混合淋巴细胞反应和增殖抑制试验,IL-10和TGF-β1的测定,与未加雷帕霉素对照组进行比较,探讨雷帕霉素在体外对CD4+CD25+Foxp3+T细胞扩增的影响。论文的第二部分,按照成人给药剂量,给实验小鼠雷帕霉素灌胃3周后,流式细胞仪检测小鼠脾细胞中CD4+CD25+T细胞的含量,实时定量PCR检测脾细胞Foxp3 mRNA的表达,磁珠分选CD4+CD25+T细胞,进行体外抑制试验,ELISA检测小鼠血清中IL-10和TGF-β1的含量,与正常未给药组小鼠比较,了解雷帕霉素在小鼠体内对CD4+CD25+Foxp3+T细胞增殖的影响。在第三部分中,通过小鼠GVHD移植模型,观察发生GVHD时小鼠体内CD4+CD25+T细胞以及脾细胞内Foxp3 mRNA表达的变化,与正常未移植和自体移植小鼠对比,明确GVHD与CD4+CD25+Foxp3+T细胞之间的相关性。在论文的第四部分,通过尾静脉和腹腔注射两种途径,建立可移植性EL9611红白血病BalB/C和CB6F1小鼠模型,外周血涂片瑞氏-吉姆萨染色以及脏器的病理检查,了解接种EL9611红白血病小鼠脾细胞后,BalB/C和CB6F1小鼠发病规律。在第五部分中,通过对发病的CB6F1小鼠进行骨髓移植,观察输注雷帕霉素体外培养扩增的CD4+CD25+Foxp3+T细胞小鼠植入、GVHD、以及白血病复发,与自体移植组和GVHD组小鼠对比,明确CD4+CD25+Foxp3+T细胞对GVHD、植入和GVL方面的影响。在论文的最后部分中,采用高分辨的方法,对21例接受单倍型相合的造血干细胞移植的供受者的HLA-Cw位点进行检测,结合临床资料,分析供受者间HLA-Cw相容性对GVHD、白血病复发以及生存的影响。【结果】(一)在上述培养体系中,CD4+T细胞培养3周后,CD4+CD25+T细胞达(76.05±2.73)%,高于未加雷帕霉素组(52.17±1.36)%(P<0.001),Foxp3 mRNA的表达是对照组的5倍(P<0.001),增殖能力是对照组的0.29倍(P<0.001),对CD4+T细胞增殖抑制能力是对照组的3.6倍(P<0.001),培养上清中IL-10和TGF-β1分别是未加雷帕霉素组的1.8倍和1.6倍(P<0.001)。(二)雷帕霉素灌胃小鼠脾细胞CD4+CD25+T细胞是对照组的2.8倍(P<0.001),Foxp3 mRNA的表达是对照组的6倍(P<0.001),磁珠分选CD4+CD25+T细胞对CD4+T细胞增殖的抑制率与对照组相比无差异(57.03% vs 56.95%)(P>0.05)。雷帕霉素组和未给药组小鼠血清中IL-10的含量分别为(166.28±24.07)pg/ml和(79.50±7.68)pg/ml,TGF-β1的含量分别为(166.28±24.07)pg/ml和(88.52±10.06)pg/ml,各组间差异有显著性(P<0.001)。(三)在发生GVHD的时间点时,自体移植组和GVHD组小鼠脾脏CD4+CD25+T细胞为15.4%,GVHD组小鼠脾脏CD4+CD25+T细胞为24.03%,而正常未移植小鼠脾脏CD4+CD25+T细胞为8.46%,各组间差异有显著性(P<0.001);Foxp3 mRNA的表达在自体移植组和正常未移植组无差异(与HPRT标化后为0.1144 vs 0.1294)(P>0.05),而GVHD组小鼠Foxp3 mRNA的表达经标化后仅为0.0331(P<0.001)。(四)8～10周龄的BalB/C和CB6F1小鼠尾静脉注射1×106 EL9611红白血病小鼠脾细胞,外周血出现白血病细胞的时间分别为接种后的第6天和第7天,死亡时间分别为13.3天和21.7天。腹腔接种1×106 EL9611红白血病小鼠脾细胞,外周血出现白血病细胞的时间分别为接种后的第17.2天和第24.6天,死亡时间分别为21.7天和27.5天。(五)Treg移植组小鼠GVHD的累积发生率为28.6%,GVHD对照组累计发生率为100%,经Log Rank检验,P=0.000,自体移植组白血病复发率为57.14%,Treg移植组未发现白血病复发。在移植后第10天、20天和30天,Treg移植组小鼠测得的嵌合度均高于GVHD组小鼠。(六)在HLA-Cw位点相合组,II～IV度aGVHD的累积发生率为77%,而在HLA-Cw位点不相合组,II～IV度aGVHD的累积发生率只有14.3%,明显低于相合组,差异有显著性(P<0.01);HLA-Cw不合组28个月累积生存率为85.7%,相合组28个月累积生存率为49%,差异无显著性(P>0.05),可能与病例数较少有关。【结论】(一)雷帕霉素在体内和体外都能促进CD4+CD25+Foxp3+T调节细胞的增殖,而且增殖的CD4+CD25+Foxp3+T调节细胞仍具有免疫调节作用。(二)发生GVHD时,CD4+CD25+T细胞增高,而CD4+CD25+Foxp3+T调节细胞的数量是降低的。(三)给CB6F1小鼠尾静脉注射1×106可移植性BalB/C小鼠EL9611红白血病脾细胞,可以建立稳定可靠的CB6F1小鼠白血病模型,但以尾静脉接种的EL9611红白血病小鼠适合于观察白血病小鼠的骨髓移植模型中GVL效应。(四)CD4+CD25+Foxp3+T调节细胞不但能有效的减轻小鼠移植GVHD的发生,同时能促进植入,不削弱GVL效应。(五)在单倍型相合的造血干细胞移植中,供受者间HLA-Cw位点的检测可以作为优化供体选择的指标之一。更多还原

【Abstract】 ObjectiveEstablish a stable system of expanding CD4+CD25+Foxp3+ regulatory T cells in vitro, investigate the relationship between CD4+CD25+Foxp3+ regulatory T cells and haploidentical GVHD, and explore the effect of the CD4+CD25+Foxp3+ regulatory T cells on engraftment, GVHD, GVL and long-term survival. Explore the role of HLA-Cw loci in haploidentical haemopoietic stem cell transplantation; afford a useful parameter in dornor selection.MethordsIn the first part, CD4+T cells derived from BalB/C mice spleen were selected by Mini MACS, and cultured in 24 wells plate for 3 weeks, which coated with 2ug/mlαCD3 monoclonal antibody, the medium system included 1ug/mlαCD28 monoclonal antibody, rhIL-2 100U/ml and 10nM RAPA (Rapamycin) with RPMI 1640 medium and 15% FBS, under the 37oC, 5%CO2 and saturated humidity incubator. After 3 weeks, CD4+CD25+T cells were detected by FCM, CD4+CD25+T cells were sorted by Mini MASC, the expression of Foxp3 mRNA was measured with real-time RT-PCR, MLR and proliferation inhibition were used to exam the function of CD4+CD25+T cells, IL-10 and TGF-β1 of supernatant were measured by ELISA, by comparing with CD4+CD25+T cells expanded without RAPA in vitro, to explore the effect of RAPA on expanded CD4+CD25+Foxp3+T cells.RAPA was administrated to Balb/C mice 0.4mg/day intragastrically as the dose of person, the control group mice of same age and weight were given with sterile water in the second part. After three weeks, CD4+CD25+T cells of splenocyte were detected by FCM; the relative levels of Foxp3 mRNA were determined by real-time quantitative RT-PCR in total splenocytes. CD4+CD25+T cells were sorted by Mini MACS for examination of inhibition, levers of IL-10 and TGF-β1 of blood serum were assessed by ELISA, the results were compared to the control group, to probe the effect of RAPA on CD4+CD25+ Foxp3+T cells proliferations in vivo.In the third part, the amount of CD4+CD25+ T cells and Foxp3 mRNA were examined among the GVHD group, autotransplantation and nontransplantation group, to identify the correlation of GVHD and CD4+CD25+Foxp3+T cells.The transferability EL9611 erythroleukemia mice model was established via injecting spleen cells of mice with EL9611 erythroleukemia into BalB/C and CB6F1 mice caudal vein and cavum abdominis in the fourth part, to learn the proceeding of EL9611 erythroleukemia in BalB/C and CB6F1 mice.In the fifth part, CB6F1 mice with EL9611 erythroleukemia were performed haploidentical bone marrow transplantation with or without CD4+CD25+Foxp3+T cells, The development of GVHD, engraftment, and leukemia relapse were observed to investigate the effect of CD4+CD25+Foxp3+T cells on GVHD, engraftment and GVL.In the last part, HLA-Cw of donors and recipients were detected by PCR-SSP, the effect of HLA-Cw in haploidentical hematopoietic stem cell transplantation was analyzed by collecting the clinical data, including incidence of GVHD, leukemia replase and long- term survival.ResultsCultured in these medium for 3 weeks, CD4+CD25+T cells developed from CD4+T cells rose from 8.84% to (76.05±2.73)%, higher than control group(52.17±1.36)%(P<0.001), Real-time quantitative RT-PCR showed that the levels of Foxp3 mRNA of CD4+CD25+T cells in RAPA group was 5 folds higher than the group without RAPA(P<0.001), the ability of proliferation was 0.29 folds higher than the control group(P<0.001), the ability of inhibition to CD4+T cells was 3.6 folds higher than the control group(P<0.001), the level of IL-10 and TGF-β1 of supernatant was 1.8 and 1.6 folds higher than control group, respectively (P<0.001).The CD4+CD25+T cell of experimental mice splenocytes was (24.13±10.06) %, while control group was (8.48±3.19) % (P<0.001). Real-time quantitative RT-PCR showed that the levels of foxp3 mRNA of experimental mice splenocytes was 6 folds higher than the control group (P<0.001). CD4+CD25+T cells sorted from experimental mice and control mice splenocytes inhibited CD4+T cells (57.03±3.63) % and (56.95±1.93) % respectively (P>0.05). The levels of IL-10 and TGF-β1 of blood serum were(166.28±24.07)pg/ml and (165.27±22.95)pg/ml in experimental group, while (79.50±7.68)pg/ml and (88.52±10.06) pg/ml in control group (P<0.001).CD4+CD25+T cells were detected by FCM at the point of GVHD development; the proportion of CD4+CD25+T cells was 15.4% and 24.03% in the autotransplantation group and allotransplantation group, respectively. But the proportion of CD4+CD25+T cells was 8.46% in nontransplantation group, the levels of Foxp3 mRNA of allotransplantation group mice were lower than autotransplantation or nontransplantation group significantly (P<0.001).1×106 EL9611 erythroleukemia cells were injected into BalB/C and CB6F1 mice (8 to 10 weeks) caudal vein and cavum abdominis. The time of leukemia cell appearance in peripheral blood was at day 6 and 7, and the mice were dead at day 13.3 and 21.7 respectively. The time of erythroleukemia cell appearance in peripheral blood was at day 17.2 and 24.6 when 1×106 EL9611 erythroleukemia cells were injected into BalB/C and CB6F1 mice cavum abdominis, but the mice were dead at day 21.7 and 27.5.The cumulative incidence of GVHD was 28.6% and 100% in Treg transplantation group and control group respectively. 4/7(57.14%) relapsed in autotranplantation group, but no one in Treg transplantation group.In haploidentical HSCT for person, The cumulative incidence of grades II–IV acute GVHD were 76.9% in HLA-Cw matched group and 14.3% in HLA-Cw mismatched group(P<0.05), 28 months disease-free survival probabilities was 46.2% in HLA-Cw matched group, and 85.7% in HLA-Cw mismatched group(P>0.05).ConclusionRAPA can promote CD4+CD25+Foxp3+T cells proliferation in vitro and vivo. The proporation of CD4+CD25+T cells could rise while CD4+CD25+Foxp3+T cells were reduced when GVHD happened. The CB6F1 EL9611 erythroleukemia mice model established by injected EL9611 erythroleukemia cells into caudal vein was reliable. CD4+CD25+Foxp3+T cells could reduce GVHD effectively, while not impair GVL. HLA-Cw mismatched in donor and receipt of haploidentical SCT for person was benefited to reducing II-IV aGVHD, and it was in favor of long-term survival. HLA-Cw might be one of the optimized parameter in dornor selection.更多还原