A novel approach to human leukocyte antigen-mismatched transplantation in patients with malignant hematological disease

Background Many patients requiring allogeneic hematopoietic stem cell transplantation (HSCT) do not have an human Ioukocyte antigen (HLA)-matched donor. Alternative donors, such as HLA mismatched family donors, are associated with higher rates of graft rejection and acute graft versus host disease (aGVHD) if T cells are not first depleted. We developed a new technique for HLA mismatched allogeneic HSCT using G-CSF primed bone marrow plus G-CSF-mobilized peripheral blood stem cells without ex vivo T cell depletion.Methods In this study, 58 patients, including 33 with high-risk or advanced leukemia, were transplanted with cells from an HLA-haploidentical family donor with 1-3 mismatched loci. After conditioning, patients received G-CSF-primed bone marrow grafts that had not been depleted ex vivo of T cells, in combination with G-CSF-mobilized peripheral blood stem cells, as well as GVHD prophylaxis.Result All patients achieved sustained, full donor-type engraftment. The incidence of grade Ⅱ-Ⅳ aGVHD was 37. 9%, including 3 patients with grade Ⅲ-Ⅳ aGVHD. The development of aGVHD was not associated with the extent of HLA disparity. Chronic GVHD was observed in 30 of 51 evaluable patients (65.4%). Fourteen patients died among whom 7 died of recurrent disease and 7 of transplant-related complications. Forty-four of the 58 patients survived, and 42 remained disease free at the time of a median follow-up of 12 months (3. 5 to 39. 5 months). The 2-year probabilities of disease-free survival were 74. 8% and 69. 3% for standard- and high-risk patients, respectively.Conclusion We developed a new method to use bone marrow from haploidentical family donors without ex vivo T cell depletion, in combination with G-PBSCs, as a source of stem cells even in cases of HLA mismatched transplantation.     

粒细胞集落刺激因子动员的异基因骨髓与外周血干细胞混合移植治疗血液系统恶性疾病

目的　探讨粒细胞集落刺激因子 (G CSF)动员的异基因骨髓与外周血干细胞混合移植治疗血液系统疾病的可行性与疗效。方法　供者给予G CSF连续 4～ 6天皮下注射 ,注射后第 3天从髂后上嵴抽取G CSF动员的骨髓血 ,第 4天开始取外周血干细胞。并立即输给受者——— 14例血液系统恶性疾病患者 (恶性肿瘤 10例 ,重症再生障碍性贫血 4例 )接受了G CSF动员的异基因骨髓与外周血干细胞混合移植 ,分析其植活率、植活速度以及急性、慢性移植物抗宿主病 (aGVHD ,cGVHD)的发生率。结果　 14例患者均获得异体植活 ,白细胞数恢复至≥ 1× 10 9/L ,血小板≥ 2 0× 10 9/L的中位时间分别为移植后的 14d(12～ 18d)和 13d (11～ 6 5d)。有 4例发生了Ⅰ度aGVHD ,3例发生了Ⅱ度aGVHD ,但无一例重度aGVHD发生。在可评估的 9例患者中有 3例出现了广泛的cGVHD。有 2例患者因白血病复发而死亡 ,另 1例因植活不良 ,再次输注供者外周血干细胞 (PBSC)后出现IV度aGVHD死亡 ,其余 11例中位随访时间 4 5 5d(84～ 715d) ,均无病生存。结论　G CSF动员的异基因骨髓与外周血干细胞混合移植可以用于血液系统恶性疾病的治疗 ,能保证持久、稳定的植活 ,植活速度快 ,急、慢性GVHD的发生率与异基因骨髓移植相比并没有增加。     

异基因造血干细胞移植患者外周血CD4+ T细胞中STAT3启动子区 DNA甲基化水平与急性移植物抗宿主病的关系

目的：探讨异基因造血干细胞移植患者外周血CD4+ T细胞中STAT3启动子区DNA甲基化水平与急性移植 物抗宿主病(acute graft versus host disease,aGVHD)的关系。方法：收集行同胞全相合异基因造血干细胞移植的40例患 者的血液样本,ELISA检测各组患者血清IL-10,TGF-β1,IL-17A,IL-17F等细胞因子水平;实时定量PCR检测各组患 者外周血CD4+ T细胞中Treg(Foxp3,CTLA4,IL-10,TGF-β1)和Th 17(RORγt,IL-17A,IL-17F)相关基因的转录水平; 实时定量PCR和Western印迹检测各组患者STAT3的表达水平;亚硫酸氢盐处理后测序(bisulfite sequencing PCR,BSP) 法检测各组患者STAT3基因启动子区DNA甲基化水平。结果：与未发生aGVHD患者比较,aGVHD患者血清中IL-10 及TGF-β1水平明显降低,IL-17A及IL-17F水平明显升高;aGVHD患者外周血CD4+ T 细胞中Foxp3,CTLA4,IL-10, TGF-β1转录水平明显降低,RORγt,IL-17A,IL-17F转录水平明显升高;aGVHD患者外周血CD4+ T细胞中STAT3的表 达水平明显升高,STAT3启动子区DNA甲基化水平明显降低,且STAT3表达水平与其启动子区DNA甲基化水平呈明显 负相关。结论：Treg/Th17的比例失衡是异基因造血干细胞移植后患者发生aGVHD的重要因素,STAT3启动子区DNA 低甲基化可能介导STAT3的过度表达,参与Treg/Th 17的比例失衡。     

甲氨蝶呤治疗异基因造血干细胞移植后的移植物抗宿主病

目的评价小剂量甲氨蝶呤治疗异基因造血干细胞移植(Allo-HSCT)后移植物抗宿主病(GVHD)的疗效。方法对31例Ⅰ—Ⅱ度急性GVHD(aGVHD)、慢性GVHD(cGVHD))和供者淋巴细胞输注后(post-DLI)GVHD患者予以甲氨蝶呤5或10mg,静脉点滴,每5～7d1次,直至GVHD症状消失／缓解、判断甲氨蝶呤无效或毒副作用不能耐受。结果aGVHD组、cGVHD组和post-DLIGVHD组总有效率分别为94％、75％和100％。皮肤型、肠道型、肝脏型、口腔型和眼型的总有效率分别为100％、60％、71％、75％和100％。甲氨蝶呤相关的毒副作用均可耐受。本治疗可减少其他免疫抑制剂的用量。结论小剂量、短期内应用甲氨蝶呤可以安全有效地治疗Allo-HSCT后的Ⅰ-Ⅱ度aGVHD、cGVHD和post-DLIGVHD。     

IL－2和IL－6基因在急性移植物抗宿主病患者外周血单个核细胞中的表达

目的：研究ＩＬ－２及ＩＬ－６在急性ＧＶＨＤ中的表达状态。方法：采用细胞原位杂交方法，对骨髓移植后发生急性ＧＶＨＤ患者的外周血单个核细胞检测其ＩＬ－２和ＩＬ－６的表达水平，测定积分光密度值。结果：ＩＬ－２表达在急性期与缓解后比较，ｔ＝５．９９，Ｐ＜０．００１，差异非常显著；ＩＬ－６表达在急性期与缓解后比较，ｔ＝３．５９，Ｐ＜０．０１，差异显著。结论：ＩＬ－２和ＩＬ－６在急性ＧＶＨＤ时表达升高，在缓解后降低，其表达状态有助于探讨ＧＶＨＤ的免疫病理损害机理。     

异基因外周造血干细胞移植治疗儿童重型再生障碍性贫血

目的探讨异基因外周血造血干细胞移植(allo—PBSCT)对儿童重型再生障碍性贫血(SAA)的疗效。方法8例接受allo—PBSCT治疗的SAA 患儿均为血缘供体移植,男6例,女2例,7～14岁,中位年龄为11岁。4例患儿为急性SAA I型,其中2例移植前合并重型感染且未能完全控制;另4例患儿为SAA Ⅱ型,患儿移植前大多经包括环孢菌素A(CSA)在内的多种治疗无效。诊断至移植的时间为1～45个月,中位时间为13个月。预处理方案由低剂量环磷酰胺（CTX）和抗淋巴细胞球蛋白（ALG）或抗胸腺细胞球蛋白（ATG）组成,采用CsA+短程甲氨蝶呤(MTX)方案预防移植物抗宿主病(GVHD)。以DNA短串联重复序列多态性分析(STR PCR)检测学植入依据。结果患者移植后均获造血重建,中性粒细胞绝对值≥0.5×109／L和血小板计数≥20×109／L的中位时间分别为移植后+12.75d(+10～+15d)和+19.88d(+15～+32d)。8例患儿均为完全供体型植入,其中1例移植后10个月出现外周血白细胞、红细胞、血小板计数逐渐下降,STR PCR显示移植排斥,经ATG、CsA 等药物治疗逐渐恢复自身造血。1例患儿出现Ⅱ度急性GVHD,1例出现局限性慢性GVHD。随访15～50个月,中位数为29个月,8例SAA患儿均存活。结论采用以免疫抑制为主的ATG/ALG+CTX预处理方案进行allo—PBSCT能够有效治疗儿童SAA,移植相关并发症少,值得临床推广应用。     

异基因造血干细胞移植术后CD4+T细胞中缺乏SIRT1诱发急性移植物抗宿主病

目的：研究异基因造血干细胞移植患者外周血CD4+T细胞中SIRT1基因表达水平与急性移植物抗宿主病 (acute graft-versus-host disease,aGVHD)的关系。方法：收集40例行同胞全相合异基因造血干细胞移植患者的血液样 本,采用实时定量PCR和Western印迹检测各组患者SIRT1的表达水平;采用Western印迹检测各组患者STAT3乙酰化 及磷酸化水平;采用免疫共沉淀和Western印迹检测各组患者SIRT1和STAT3结合水平;aGVHD患者CD4+T细胞过表 达SIRT1后采用Western印迹检测STAT3乙酰化及磷酸化水平,采用实时定量PCR检测Th17相关基因RORγt,IL-17A, IL-17F的表达。结果：与未发生aGVHD患者比较,aGVHD患者外周血CD4+T细胞中SIRT1表达水平明显降低;STAT3 表达水平、乙酰化及磷酸化水平均明显升高,且STAT3乙酰化与磷酸化水平呈明显正相关(r=0.69,P<0.01);STAT3 与SIRT1结合显著减少。aGVHD患者外周血CD4+T细胞中过表达SIRT1后,STAT3乙酰化及磷酸化水平均明显降低, RORγt,IL-17A,IL-17F的表达明显减少(P<0.05)。结论：CD4+T细胞中SIRT1表达不足是异基因造血干细胞移植术后 患者STAT3高度乙酰化及磷酸化,介导Th 17的相关基因表达升高,进而诱发aGVHD的重要因素。     

异基因造血干细胞移植后aGVHD与调节性T细胞、IL-35的相关性

目的   探索异基因造血干细胞移植（allo-HSCT）后发生急性移植物抗宿主病（aGVHD）的患者外周血中调节性T细胞及其相关转录因子、细胞因子的水平,以期获得能够有效预测患者发生aGVHD的生物学指标。方法   外周血样品采自接受造血干细胞移植术的患者和健康人,其中aGVHD阳性组13例,aGVHD阴性组10例,正常对照组21例。采用流式细胞术检测各组外周血中CD4+CD25+Foxp3+Treg 细胞占CD4+T淋巴细胞的百分比,应用实时定量PCR检测外周血单个核细胞中Foxp3转录因子的相对表达量,应用酶联免疫吸附试验检测外周血血浆中Treg相关细胞因子IL-35的表达水平。结果   aGVHD阳性组外周血CD4+CD25+Foxp3+Treg细胞百分比、单个核细胞中Foxp3转录因子的相对表达量、血浆中IL-35的表达水平较aGVHD阴性组和正常对照组明显降低（P<0.05）,且经有效治疗后各指标显著升高。在aGVHD阳性组中CD4+CD25+Foxp3+Treg细胞百分比、Foxp3的相对表达量与aGVHD的危险度分级呈负相关（P<0.05）。配对样本资料分析显示,aGVHD发生时外周血CD4+CD25+Foxp3+Treg细胞百分比、Foxp3 mRNA的相对表达量与aGVHD发生前1~2周相比明显下降（P<0.05）。结论   异基因造血干细胞移植后患者发生aGVHD时CD4+CD25+Foxp3+Treg细胞的百分比、其转录因子的表达量及IL-35水平均发生明显降低,且在治疗后明显升高,可以为临床预测aGVHD的发生发展及预后提供理论依据和实验基础。     

HLA配型不合情况下造血干细胞移植的新方法

目的:采用新方法进行非体外去除T细胞的人类白细胞抗原(human leukocyte antigen, HLA)配型不合造血干细胞移植.方法:58例血液恶性肿瘤患者,33例为高危或难治复发白血病,接受了至少一个HLA位点不合的家庭供者造血干细胞移植.移植物为经粒细胞集落刺激因子(granulocyte clony-stimulating factor, G-CSF)动员的骨髓以及外周血造血干细胞,而无需体外去除T细胞.移植物抗宿主病(graft-versus-host-disease, GVHD)预防采用环孢菌素A 霉酚酸酯 短程甲氨喋呤方案.结果:所有患者均获得持久、完全供者植入.58例患者中发生Ⅱ度及以上急性GVHD 22例(37.9%),其中Ⅲ度和Ⅳ度急性GVHD分别为2例和1例,其严重程度与HLA不合程度无相关;42例可评估患者中,慢性GVHD为26例(61.9%),广泛型和局限型分别为11例和15例.复发9例,除1例外均为复发、难治白血病患者.死亡14例,其中7例死于疾病复发,另7例死于移植相关合并症,其中严重感染和间质性肺炎各2例,巨细胞病毒性脑炎、病毒型肝炎和急性GVHD死亡各1例.中位随访10月(2～37.5月),58例患者中42例无病存活(disease-free survival, DFS),高危患者2年DFS明显低于标危患者,分别为63.2%和77.6%(P= 0.04).DFS与供受者间HLA配型不合程度、急性GVHD严重程度及回输干细胞数量无关.结论:(1)无需体外去除T细胞的新方法可克服HLA屏障、安全有效地用于HLA不合的移植;(2)G-CSF动员的外周血干细胞可安全用于HLA不合的造血干细胞移植.     

阻断4-BB/4-1BB配体信号通路对抑制大鼠肝脏移植免疫排斥反应的实验研究

Background Blocking the 4-1BB/4-1BB ligand （4-1BBL） signal may modulate the secretion of Th1/Th2 cytokines and prolong the survival of the grafts, which play a key role in organ transplantation tolerance. The aim of this study was to investigate the role of blockade of the 4-1BB/4-1BBL co-stimulatory pathway with 4-1BBL monoclonal antibody （mAB） in acute rejection of rat orthotopic liver transplantation. Methods The orthotopic liver transplantation model was set up, while male Lewis rats were used as liver donors and Brown-Norway rats as recipients. The recipient rats were intravenously injected with anti 4-1BBL mAB or isotype control antibody. Groups were monitored for graft survival after transplantation. Plasma chemistry, including aspartate transaminase （AST）, alanine aminotransferase （ALT）, and bilirubin （BIL）, was assayed. The concentrations of interleukin （IL）-2, IL-10 and interferon （IFN）-γ in plasma were also measured by enzyme-linked immunosorbent assay. Allograft histology images were collected under light microscope and electron microscope. Results Isotype antibody treated recipients exhibited elevated plasma levels of liver injury markers including AST, ALT and BIL, progressive portal and venous inflammation and cellular infiltration of the liver ailografts, and a mean graft survival time （MST） of 10.9 days. Administration of anti 4-1BBL mAB resulted in a decrease in plasma levels of liver injury markers and the concentrations of IL-2, IL-10 and IFN-γ. The histological grade of rejection on day 7 decreased and MST （17.3 days） increased substantially. Conclusions These results demonstrate that attenuation of acute rejection follows the blockade of the 4-1BB/4-1BBL co-stimulatory pathway with 4-1BBL monoclonal antibody and strongly suggest it is a promising strategy to prevent progression of graft rejection by suppressing T cell-mediated immunity.     

亲缘间HLA半相合外周血造血干细胞移植治疗急性白血病的临床研究

目的探讨HLA半相合外周血造血干细胞移植（PBSCT）治疗急性白血病的疗效及安全性。方法4例急性白血病患者行HLA不全相合PBSCT,其中急性淋巴细胞白血病2例,急性非淋巴细胞白血病-M4b1例、M4EO1例,移植时均处于完全缓解状态。HLA配型1、2个位点不合各1例,3个位点不合2例。预处理方案由全身照射或白消胺、阿糖胞苷、环磷酰胺、司莫司汀组成。移植物抗宿主病（GVHD）的预防采用环胞素A、甲氨蝶呤、霉酚酸酯、抗胸腺细胞球蛋白四联方案。结果患者移植过程顺利,4例患者均获得造血功能重建并达完全供者植人。3例出现Ⅰ度急性GVHD,1例出现Ⅱ度急性GVHD。1例并发巨细胞病毒间质性肺炎,2例并发真菌肺炎。至今2例患者分别无病存活30月和15月,1例目前为移植后4月,1例死亡。结论HLA半相合PBSCT疗效较好、安全可行,为无HLA完全相合供者的白血病患者提供了新的治疗手段。     

定量监测Janus激酶3基因变化在移植物抗宿主病中的初步研究

目的 观察异基因造血干细胞移植（allo—HSCT）后外周血单个核细胞Janus激酶3（JAK3）mRNA表达水平的变化,探讨其与移植物抗宿主病（GVHD）之间的关系。方法 采用荧光定量逆转录聚合酶链反应（FQ—RT—PCR）技术连续监测26例allo—HSCT后不同时问83份外周血单个核细胞JAK3 mRNA表达水平。结果 26例进行allo—HSCT的患者,14例患者移植后未发生GVHD,JAK3／ABL mRNA表达水平中位数为8．71;1例发生急性GVHD（aGVHD）,JAK3／ABL为31．94;11例发生慢性GVHD（cGVHD）,JAK3／ABL为19．23。连续观察4例患者,移植后1个月的JAK3／ABL表达水平与移植前比较无变化,但随着GVHD的出现,JAK3／ABL表达水平又很快升高,并且随着GVHD治疗好转,JAK3／ABL表达水平又很快下降。结论 GVHD患者JAK3 mRNA表达水平与GVHD的病情演变相关。     

Modified conditioning regimen busulfan-cyclophosphamide followed by allogeneic stem cell transplantation in patients with multiple myeloma

Background Allogeneic stem cell transplantation is a potential curative approach in patients with multiple myeloma. The very high transplant related mortality associated with standard allogeneic stem cell transplantation is currently the major limitation to wider use of this potentially curative treatment modality. The challenge for clinical investigators is to reduce the incidence of post-transplant complications for patients receiving autologous hematopoietic stem cell transplantion for multiple myeloma. In this study the toxicity and efficacy of modified myeloablative conditioning regimen followed by allogeneic stem cell transplantation was investigated in patients with multiple myeloma. Methods The conditioning regimen consisted of hydroxyurea, cytarabine, busulfan, cyclophosphamide, and semustine. Ten patients underwent allogeneic transplantation among them hydroxyurea （40 mg/kg） was administered twice on day -10 and cytarabine （2 g/ms） was given on day -9, busulfan was administered orally in four divided doses daily for 3 days （days -8 to -6）. The dose of busulfan was 12 mg/kg in the protocol followed by cyclophosphamide intravenously over 1 hour on days -5 and -4 （1.8 g/m^2）, and with semustine （Me-CCNU） 250 mg/m^2 on day -3. Results Chimerism data were available on all patients and all patients achieved full donor chimerism without graft failure. Six patients had not acute graft-versus-host disease （GVHD, 36.4%; 95% CI： 13.9%-38.6%）. Two patients （18.2%） developed grade Ⅰ acute GVHD （95% CI： 10.9%-35.9%） and grade Ⅱ acute GVHD occurred in one patient （9.1%; 95% Cl. 8.4%-32.3%）. Severe grade IVa GVHD was seen in one patient, who died from acute GVHD. The incidence of chronic GVHD was 22.2% （95% Cl： 11.7%-36.7%）, among them one died of severe grade IV GVHD and one developed multiorgan failure on day ＋170; the treatment-related mortality was 22.0% （95% Cl： 10.3%-34.1%）. The overall 4-year survival rate was 67.8% （95% Cl： 16.3%-46.7%）. The estimated 4-year progression-free survival rate was 58.5% （95% CI： 13.7%-41.8%）. The 4-year complete remission was 72.7% （95% CI： 27.8%-49.6%）. One patient relapsed after 4 months and achived the complete remission after receiving the donor lymphocyte infusion. Conclusions Modified conditioning regimen busulfan-cyclophosphamide with peripheral blood stem cells＋bone marrow cells transplantation result in a low incidence of severe GVHD with a relatively low treatment-related mortality, high complete remission rates and a long-term survival.     

环孢素A＋甲氨蝶呤＋霉酚酸酯＋抗胸腺细胞球蛋白四联方案预防无关供体造血干细胞移植中的移植物抗宿主病

OBJECTIVE: To evaluate the efficacy of quadruple therapy with cyclosporine (CsA), methotrexate (MTX), mycophenolate mofetil (MMF) and low-dose antithymocyte globulin (ATG) for graft-versus-host disease (GVHD) prophylaxis in unrelated donor hematopoietic stem cell transplantation (URD-HSCT). METHODS: Thirteen patients with leukemia received URD-HSCT, of whom 11 had HLA genotypes and 2 had mismatch for 1 genetic locus. Another 11 leukemia patients all serologically matched underwent related donor (RD)-HSCT. Total body irradiation (TBI) plus cyclophosphamide (CTX) was adopted in 19 cases and modified BuCY conditioning regimen (hydroxyurea, busulfan, Ara-C, Cyclophosphamide ) in the other 5 cases. All the patients received CsA+MTX protocal for GVHD prophylaxis, and in those undergoing URD-HSCT, additional MMF and low-dose ATG were used. RESULTS: The incidence and severity of regimen-related toxicity differed little between unrelated and related transplantation. Acute GVHD (aGVHD) occurred in 46.2% of the patients undergoing URD- HSCT and in 55.6% of those with RD-HSCT, respectively. Four patients had chronic GVHD (cGVHD), in the 7 ones who could be followed up after URD-HSCT; 6 of the 9 patients with RD-HSCT developed cGVHD postoperatively. One patient with URD-HSCT died of hemorrhagic cystitis and another with RD-HSCT died of cytomegalovirus (CMV) pneumonia. The at one-year disease-free survival rate was 87.5% and 90.9% in patients with unrelated and related transplantation respectively. Significant difference was not noted in the positivity rate of CMV antigen between the patients receiving URD-HSCT or RD-HSCT. CONCLUSION: CsA+MTX in combination with MMF and low-dose ATG may decrease the incidence and severity of aGVHD without increasing transplant-related mortality or CMV infection.     

双向诱导H─2不同小鼠供受者间免疫耐受进行骨髓移植

目的：预防骨髓移植后发生移植物抗宿主病和排斥反应。方法：紫外线Ｂ体内照射、紫外线Ｂ照射的供（受）者特异输血及环孢菌素Ａ结合的方式、在供者（Ｃ５７ＢＬ／６）、受者（Ｂａｌｂ／ｃ）小鼠间行双向诱导免疫耐受，再进行骨髓移植。结果：１只小鼠在移植后第３０ｄ死于移植物抗宿主病，９／１０的小鼠存活＞２００ｄ。而混合移植（Ｂａｌｂ／ｃ＋Ｃ５７ＢＬ／ｃ→Ｂａｌｂ／ｃ，１：４）组，１０／１０的小鼠存活＞２００ｄ。结论：双向诱导免疫耐受预防小鼠骨髓移植后发生移植物抗宿主病和排斥反应是必要的。     

HMGB1/GADD45A介导急性移植物抗宿主病患者CD4+ T细胞STAT3启动子的去甲基化

目的：研究急性移植物抗宿主病(acute graft-versus-host disease,aGVHD)患者CD4+ T细胞中STAT3基因启动 子DNA病理性低甲基化的分子调控机制。方法：收集行同胞全相合异基因造血干细胞移植的42例患者的血液样本。 采用实时定量PCR和Western印迹检测各组患者生长阻滞与DNA损伤诱导蛋白45A(growth arrest and DNA damage-inducible protein 45 alpha,GADD45A)的表达水平,免疫共沉淀检测各组患者高迁移率族蛋白1(high mobility group box-1 protein, HMGB1)与GADD45A的结合水平,染色质免疫共沉淀检测各组患者HMGB1和GADD45A与STAT3启动子的结合水 平;正常CD4+ T细胞中过表达HMGB1及干扰GADD45A表达后用Western印迹检测STAT3表达水平,亚硫酸盐测序检测 STAT3启动子区DNA甲基化水平。结果：与未发生aGVHD患者比较,aGVHD患者外周血CD4+ T细胞中GADD45A表达 水平明显升高;HMGB1与GADD45A的结合显著增加;HMGB1,GADD45A与STAT3启动子的结合水平均明显升高, 且STAT3启动子区HMGB1,GADD45A结合水平与其DNA甲基化水平呈高度负相关(分别r=0.719,r=0.840;P<0.01)。 与单纯过表达HMGB1相比,正常CD4+ T细胞中过表达HMGB1叠加干扰GADD45A表达后STAT3表达明显降低,STAT3 启动子DNA甲基化水平明显升高。结论：CD4+ T细胞中HMGB1和GADD45A表达升高是异基因造血干细胞移植后患者 STAT3启动子DNA低甲基化,STAT3高表达,进而诱发aGVHD的重要因素。     

Low incidence of severe aGVHD and accelerating hemopoietic reconstitution in allo-BMT using lenograstim stimulated BM cells

Objectives To investigate the efficacy of accelerating hemopoietic reconstraction and reducing a graft versus host disease (GVHD) in Allo-BMT receiving lenogras tim stimulated donor marrow and to assess the preliminary biological mechanism .Methods The donors for thirty patients (study group) with leukemia were given lenograsti m 3-4 μg·kg(-1) ·d(-1) for seven days prior to marrow harvest. The results of subsequent engraftment in the recipients was compared w ith fifteen donors without G-CSF (control group). Five donors themselves were studied t o assess the effects of lenograstion on hematopoietic progenitor cells and lym phocyte subsets in BM.Results The stimulated bone marrow contained a higher number of nucleated cells, CFU-GM and CD34(+) cells (P&lt;0.01). The hematopoetic reconstitution was accelerat ed. Until granulocyte counts exceeded 0.5×10(9) /L and plalete counts exceeded 20×10(9) /L, the days were 16.7±3.2 and 18.4±3.0 days as compared with t hose of the control group (22.5±5.1 and 26.3±5.9 days respectively, P &lt; 0.01). The incidence of grade Ⅱ-Ⅳ aGVHD was very low, only one case with g rade Ⅱ aGVHD on the skin in the study group. Four out of fifteen patients (2 6.7%) in the control group had grade Ⅱ-Ⅳ aGVHD (P&lt;0.05) . The number of T lymphocyte subsets in the harvested BM stimulated by G-CSF changed. In comp arison with the control group, CD4(+) decreased and CD8(+) increased significan tly (P&lt;0.01). The changes of progenitor cells and T lymphocyte subsets in BM from pre- to post- G-CSF stimulation indicated that the percentage o f CD4(+) cells reduced (P&lt;0.05), that of CD8(+) cells, and that of CD34(+) i ncreased (P&lt;0.01). The incidence of chronic GVHD and relapse of leukemia were not different significantly between both groups.Conclusions Allogenic bone marrow transplant (Allo-BMT) donors given G-CSF can accelerate engraftment and minimize the incidence of sever e aGVHD. There is a trend in favour of improved transplant-related complicatians.     

环孢素A+甲氨蝶呤+霉酚酸酯+抗胸腺细胞球蛋白四联方案预防无关供体造血干细胞移植中的移植物抗宿主病

目的评价环孢素A(CsA) 甲氨蝶呤(MTX) 霉酚酸酯(MMF)和低剂量抗胸腺细胞球蛋白(ATG)预防无关供体造血干细胞移植(URD-HSCT)中移植物抗宿主病(GVHD)的疗效。方法13与11例白血病患者分别接受URD-HSCT和相关供体(RD)-HSCT。移植预处理方案:19例应用全身放疗 环磷酰胺方案、5例应用改良BuCY(羟基脲、马利兰、阿糖胞苷、环磷酰胺)方案;无关移植病人供体与受体之间HLA-A、B、DR基因位点完全相合11例,1个基因位点不合2例。所有相关移植病人供体与受体之间血清学位点均为全相合;预防GVHD方案:所有病人均接受CsA MTX方案,行URD-HSCT病人在CsA MTX方案基础上加用MMF和ATG。结果移植后所有患者均获得造血重建,移植中预处理相关毒性(RRT)发生率和程度在无关与相关移植中二者无差异(P>0.05);急性GVHD(aGVHD)在无关与相关移植病人分别为46.2%和55.6%,在可统计的慢性GVHD(cGVHD)病人中,无关移植是4/7例,相关移植6/9例;无关与相关移植病人中分别有1例死于移植相关并发症,移植后1年估计无病生存率在无关与相关移植分别为87.5%和90.9%;移植后巨细胞病毒(CMV)抗原阳性率在无关与相关移植无差异(P>0.05)。结论CsA MTX MMF ATG四联方案预防URD-HSCT中GVHD能降低aGVHD的发生及其程度,不增加移植后相关死亡率和CMV感染率。     

无关供体外周血干细胞和骨髓移植治疗白血病的比较研究

目的评价无关供体外周血干细胞和骨髓移植的造血重建、T细胞重建、感染、GVHD及疗效的差异。方法46例患者接受无关供体造血干细胞移植(URD-HSCT),其中16例患者接受无关供者外周血干细胞移植(外周血组),30例患者接受无关供者骨髓移植(骨髓组)。流式细胞仪测定移植后1年内不同时间点患者的T细胞免疫重建。统计分析两组患者移植后白细胞(WBC)和血小板(BPC)重建时间,T细胞重建,感染发生率,移植物抗宿主病(GVHD)、白血病复发、无病生存(DFS)情况。结果除1例骨髓干细胞移植患者未造血重建外,其余45例均获得造血重建。外周血组和骨髓组WBC重建时间分别为 (12.81±4.15)和 (16.21±3.09)d(P=0.003);白细胞重建时间分别为 (15.50±6.91)和 (20.31±7.19)d(P=0.035),外周血组白细胞和血小板重建时间均快于骨髓组。外周血组和骨髓组患者移植后1、3、6、9、12月的T细胞重建无显著性差异。外周血组和骨髓组的移植后早期感染发生率分别为37.50%和50.00%,二者无显著性差异(P=0.644)。外周血组与骨髓组急性GVHD(aGVHD)的发生率分别为56.25%和70.00%,其中Ⅲ-Ⅳ°aGVHD的发生率在二组分别为18.75%和13.79%;在可统计的患者中,慢性GVHD(cGVHD)的发生率外周血组为30.77%(4/13),骨髓组为36.36%(8/22),aGVHD和cGVHD发生率二组比较均无差异(P值分别为0.456和0.413)。非白血病复发移植相关死亡率外周血组和骨髓组分别为18.75%和33.33%,二者无显著性差异(P=0.295)。外周血组与骨髓组移植后分别有3例和2例复发,二者的复发率无显著性差异(P=0.226);移植后2年DFS在外周血组与骨髓组分别为62.19%和56.23%,二者无显著性差异(P=0.615)。结论无关供体外周血干细胞移植后的造血重建比骨髓移植迅速,但两者间移植后T细胞重建、感染发生率、GVHD及DFS并无显著性差异。     

酶联免疫斑点法研究小鼠急性移植物抗宿主病模型中产生干扰素γ细胞的水平

目的：探讨异基因小鼠混合淋巴细胞反应（MLR）和急性移植物抗宿主病（aGVHD）模型中，供者针对受者细胞反应后产生干扰素-γ细胞（IFN-γ PC）的水平。方法：在近交系MHC不合的异基因小鼠脾细胞MLR和aGVHD模型中，用酶联免疫斑点法（ELISPOT）对供者针对受者细胞反应后IFN-γ PC的水平及其影响因素进行了研究。结果：在异基因小鼠脾细胞MLR后，用ELISPOT检测到IFN-γ PC显著升高，具有针对受者细胞的特异性。在异基因小鼠aGVHD模型中，重度aGVHD小鼠脾细胞与未移植的异基因受鼠脾细胞在MLR后，IFN-γ PC较中度aGVHD明显升高，无aGVHD药物预防组较预防组IFN-γ PC明显升高，并具有针对受者细胞的特异性，与aGVHD的临床评分相一致。结论：用ELISPOT可以快速而敏感地检测异基因小鼠脾细胞MLR中的同种异体反应，并和aGVHD的程度以及病程具有相关性。