骨髓输注减轻大鼠肺移植急性排斥反应的研究

目的 研究供体骨髓细胞输注减轻大鼠肺移植急性排斥反应。方法 应用Cuff技术改进大鼠原位单肺移植模型，同时输注供体骨髓细胞，采集受体鼠外周血动态监测嵌合；存活4周大鼠杀死后切取移植肺进行排斥反应分级，切取脾应用免疫组织化学方法检测组织嵌合。结果 流式细胞术在骨髓输注组外周血中检测到明显的嵌合，在脾脏中得到组织嵌合。骨髓输注组移植肺急性排斥反应明显减轻。结论 肺移植同时输注供体骨髓细胞，可以产生显著的供体细胞与受体细胞嵌合，有效地抑制大鼠肺移植急性排斥反应。     

Predictors of Organ Allograft Tolerance Following Hematopoietic Cell Transplantation

Using the miniature swine large animal model we have attempted to determine the relationship between tolerance and the presence of donor cells in the bone marrow, thymus and lineages of peripheral blood in a series of hematopoietic cell transplant recipients receiving delayed donor allografts without immunosuppression. Twenty-two animals receiving hematopoietic cell transplantation and a delayed organ allograft were analyzed. Assays for presence of donor CFUs in bone marrow (by PCR), thymic chimerism (by FACS and PCR/Southern Blot), peripheral blood chimerism (by FACS), and in vitro responsiveness to donor MHC were performed. Presence of donor BM CFUs, thymic chimerism and multilineage peripheral blood chimerism at the time of organ transplantation all correlated precisely with subsequent allograft tolerance (p < 0.001, p < 0.001, p < 0.005 respectively). These parameters were therefore accurate predictors (Positive Predictive Value (PPV) = 100% in all) of tolerance. In vitro assays of responsiveness were also highly associated (p < 0.002, p < 0.002 respectively), but were not as accurate predictors of subsequent organ tolerance (CML PPV = 80%). Engraftment, as indicated by the presence of donor derived CFU in the bone marrow, detectable thymic chimerism and multilineage peripheral blood chimerism are reliable predictors of subsequent donor allograft acceptance in hematopoietic cell transplant recipients.     

输注同基因骨髓间充质干细胞联合供体脾组织移植诱导肝移植大鼠免疫耐受的研究

摘要：目的 观察输注与受体同基因骨髓间充质干细胞联合供体脾组织移植对诱导大鼠肝移植术后免疫耐受作用,探讨其可能的机制。方法 实验鼠随机分为4组,每组24只。A组行DA-Lewis大鼠原位肝移植,B组行DA-Lewis大鼠原位肝移植术后予口服环孢素,C组行DA-Lewis大鼠原位肝移植同期输注Lewis大鼠骨髓间充质干细胞。D组在C组的基础上术中同期行DA大鼠脾组织移植。观察各组生存期,肝功能情况,血清细胞因子水平,嵌合体的形成情况及肝脏的病理变化。结果 与其他各组相比,D组大鼠存活时间明显延长,术后D组血清ALT、AST、TBIL较A组显著降低,与B、C组相近;B、C、D组IL-2、IFN－γ均升高,但低于A组（P<0.05）;、而IL-4和IL-10高于A组（P<0.05）,B、C组移植肝仅呈急性轻度排斥反应,A组呈急性重度排斥反应,D组未见明显排斥反应。30d后D组受体脾脏中供体阳性细胞明显高于各组（P<0.05）。结论 大鼠肝脏脾组织移植后输注与受体同基因骨髓间充质干细胞后能减轻移植肝的排斥作用,甚至诱导免疫耐受。     

异基因造血干细胞移植后早期嵌合状态研究

目的:建立多重PCR扩增短串联重复序列(STR)结合毛细管电泳,定量检测异基因造血干细胞移植(Allo-HSCT)后受者体内供、受体来源血细胞嵌合率的方法及灵敏度。并探讨该方法的早期定量检测对Allo-HSCT后白血病早期复发进行干预的指导意义。方法:将2名健康成人静脉血分离单个核细胞,并按供、受体单个核细胞不同百分比制成模拟嵌合体标本并提取基因组DNA,用多重PCR扩增后,进行毛细管电泳,确定基因位点及相应峰面积。15例Allo-HSCT患者移植前采集供、受者外周血,移植后采集受者外周血,分别提取DNA,进行PCR扩增和毛细管电泳,确定STR基因位点及峰面积,计算混合样本中2单个样本DNA含量百分比及Al-lo-HSCT患者嵌合率。结果:①当混合样本中某一细胞比例为4%以上时,即可从电泳图中明确区分混合样本中该单个样本的基因型,且扩增前细胞百分率与扩增后两者峰面积比值呈直线相关;②所有患者在移植后2周内均出现供者来源的细胞,13例患者在术后30d内均达稳定嵌合。2例患者移植后出现不稳定混合嵌合状态或复发,经干预治疗后转为完全嵌合状态。结论:STR毛细管电泳PCR方法非常敏感,能应用于Allo-HSCT后早期嵌合体的定量检测。嵌合状态的早期定量检测对了解Allo-HSCT后的移植物的植入状态和指导早期干预治疗均有重要参考价值。     

BuCy为主的低强度预处理方案异基因造血干细胞移植治疗恶性血液病

目的在高龄或同时存在不能耐受常规预处理方案的恶性血液病患者中,采用一种低强度预处理方案进行异基因造血干细胞移植治疗。方法12例患者中,急性淋巴细胞白血病(ALL)4例,慢性粒细胞白血病(CML)4例,伴原始细胞增多的难治性贫血(MDSRAEB)2例,急性髓系白血病2例。10例为HLA完全相合同胞移植,2例为1个位点不相合的同胞移植。预处理方案包括低剂量白消安2mg·kg-1·d-1,共3d,阿糖胞苷2g/m2,1～2次,环磷酰胺1.0g·m-2·d-1,共2d,抗胸腺细胞球蛋白(ATG)2.5mg·kg-1·d-1,共4d。11例患者采用经GCSF动员的供者骨髓加外周血干细胞移植,1例采用单纯外周血干细胞移植。输注有核细胞数平均为7.19×108/kg,CD34+细胞数平均为2.53×106/kg。急性移植物抗宿主病(aGVHD)预防采用环孢菌素A(CsA),短程甲氨蝶呤及霉酚酸酯联合方案。采用细胞遗传学、分子生物学及DNA短串联重复序列多态性(STR)分析方法鉴定供者干细胞植入情况。结果12例患者均能耐受此预处理方案,未发生严重预处理相关并发症。中性粒细胞绝对数>0.5×109/L的中位时间15(11～17)d,血小板植活中位时间15(10～23)d。移植后1个月鉴定植活情况,11例患者均全部转为供者型,1例患者为供、受者嵌合状态。出现aGVHD5例(41.6%),其中Ⅰ度2例,Ⅱ度2例,Ⅲ度1例,aGVHD发生率     

Proliferation of L02 human hepatocytes in tolerized genetically immunocompetent rats

AIM： To investigate whether human hepatocytes could proliferate after transplantation to normal immunocompetent rats treated with 2-acetaminofluorene or Retrorsine and partial hepatectomy. METHODS： L02 hepatocyte-tolerant Sprague-Dawley rats were injected with Retrorsine, 2-acetaminofluorene or normal saline. L02 hepatocytes were then transplanted via the spleen. Human albumin and its mRNA, specific proliferating cell nuclear antigen （PCNA）, L02 hepatocyte dynamic distribution, number density and area density of PCNA-positive cells in the liver were determined. RESULTS： All the examined indicators were not significantly different between the rats treated with 2-acetaminofluorene and normal saline, which was not the case with rats treated with Retrorsine. A dynamic distribution of L02 hepatocytes in the rat liver was detected from wk 1 to mo 6 after transplantation in the Retrorsine group and from wk 1 to 10 in the 2-acetaminofluorene group. Human albumin and its mRNA were detected from wk 2 to mo 6 in the Retrorsine group and from wk 1 to 8 in the 2-acetaminofluorene group. Specific human PCNA was detected in the rat liver from wk 2 to mo 6 in the Retrorsine group and from wk 2 to 6 in the 2-acetaminofluorene group. Human albumin and its mRNA contents as well as the number of PCNA positive cells reached a peak at wk 4. CONCLUSION： L02 human hepatocytes could not proliferate significiantly after transplantation to the normal, immunocompetent rats treated with 2-acetaminofluorene.L02 human hepatocytes can survive for 10 wk after transplantation and express human albumin for 8 wk. L02 human hepatocytes can proliferate and express human albumin for 6 mo after transplantation to the rats treated with Retrorsine. The chimeric L02 human hepatocytes, which then underwent transplantation into tolerant rats, were normal in morphogenesis, biochemistry and function.     

Treosulfan and Fludarabine Conditioning for Hematopoietic Stem Cell Transplantation in Children with Primary Immunodeficiency: UK Experience

We previously published results for 70 children who received conditioning with treosulfan and cyclophosphamide (n = 30) or fludarabine (n = 40) before undergoing hematopoietic stem cell transplantation (HSCT) for primary immunodeficiency (PID). Toxicity was lower and T cell chimerism was better in the patients receiving fludarabine, but cohort numbers were relatively small and follow-up was short. Here we report outcomes of 160 children who received homogeneous conditioning with treosulfan, fludarabine, and, in most cases, alemtuzumab (n?=?124). The median age at transplantation was 1.36 years (range, .09 to 18.25 years). Donors included 73 matched unrelated, 54 1 to 3 antigen-mismatched unrelated, 12 matched sibling, 17 other matched family, and 4 haploidentical donors. Stem cell source was peripheral blood stem cells (PBSCs) in 70, bone marrow in 49, and cord blood in 41. Median duration of follow-up was 4.3 years (range, .8 to 9.4 years). Overall survival was 83%. No patients had veno-occlusive disease. Seventy-four patients (46%) had acute GVHD, but only 14 (9%) greater than grade II. Four patients underwent successful retransplantation for graft loss or poor immune reconstitution. Another patient experienced graft rejection and died. There was no association between T cell chimerism?>95% and stem cell source, but a significant association was seen between myeloid chimerism?>95% and use of PBSCs without an increased risk of significant GVHD compared with other sources. All 11 patients with severe combined immunodeficiency diagnosed at birth were alive at up to 8.7 years of follow-up. Long-term studies are needed to determine late gonadotoxic effects, and pharmacokinetic studies are needed to identify whether specific targeting is advantageous. The combination of treosulfan, fludarabine, and alemtuzumab is associated with excellent results in HSCT for PID.     

Donor cell leukemia after allogeneic peripheral blood stem cell transplantation: a case report and literature review

A 49-year-old male developed recurrent acute myeloid leukemia 27 months after allogeneic peripheral blood stem cell transplantation (PBSCT) from an HLA-identical brother. The immunophenotype of the blastic cell population was incompatible with that of the pre-transplant blast cells; a mutation in C/EBPA gene was found in the pre-transplant blast cells that was not present in the post-transplant blast cells, and short tandem repeat analysis of marrow cells, which included 71% blasts, showed complete donor chimera. Thus, this recipient developed donor cell leukemia (DCL). The donor was healthy when DCL developed in the recipient as well as before donation of the peripheral blood stem cells. Only five cases of DCL after PBSCT have been reported in the literature. As a mechanism for the development of DCL, a vigorous proliferative demand on the donor cells, which often correlates with a higher likelihood of replication error or mutation, has been proposed. Peripheral blood stem cells might have an advantage in that they are associated with a low incidence of DCL development because PBSCT recipients receive a higher total cell dose than recipients of bone marrow or cord blood cells.     

同种异基因造血干细胞移植后基于血细胞嵌合状态的个体化免疫治疗

目的:研究同种异基因造血干细胞移植(allo-HSCT)后血细胞嵌合率变化与复发的关系;观察根据血细胞嵌合率变化给予个体化免疫抑制剂治疗和供者淋巴细胞输注(DLI)的疗效。方法:106例供者细胞顺利植入的allo-HSCT患者,采用聚合酶链反应(PCR)扩增短串联重复序列的方法,动态检测移植后T淋巴细胞、B淋巴细胞、自然杀伤(NK)细胞的嵌合率。根据血细胞嵌合率的变化调整免疫抑制剂剂量和DLI的使用。结果:6例患者在移植后2个月,供者T细胞嵌合状态一直为混合嵌合(MC),将免疫抑制剂减量后均达到完全供者嵌合(FDC)。12例患者在移植后1～5个月,发生供者T细胞嵌合率下降,予免疫抑制剂减量后转为FDC。24例患者血液学复发或髓外复发(进展),有6例在复发前共发生10例次血细胞嵌合率下降,经免疫抑制剂减量或停药后一度回升至FDC,但最终血液学或髓外复发。12例患者在复发或疾病进展后停用免疫抑制剂,共给予DLI23例次,其中8例在DLI前或后给予化疗,最终5例再次达到完全缓解,其余患者最终均因疾病复发死亡。Ⅱ度及Ⅱ度以上急性移植物抗宿主病(GVHD)发生率为28.3%。慢性GVHD发生率为55.7%。中位随访期为17(1.5～90.0)个月,无病生存65例,死亡41例。67例标危患者预期3年生存率为59.0%;39例高危患者预期3年生存率为44.7%。结论:T淋巴细胞、NK细胞和B淋巴细胞的嵌合状态可作为血液恶性肿瘤复发的预测指标;基于血细胞嵌合率的个体化免疫治疗可以推迟甚至避免临床复发,且不增加急性GVHD的发生。     

Suppressed alloreactivity and mixed chimerism in rats with accepted cardiac allografts by intrathymic injection of donor bone marrow cells

Intrathymic injection of donor bone marrow cells (ITBMCs) at the time of transplantation and treatment with antilymphocyte serum (ALS) permitted the indefinite survival of Brown Norway (BN, RT1ⁿ) rat heart grafts in 6 out of 8 Lewis (LEW, RT1^l) rat recipients. LEW recipients with long-surviving BN heart grafts (LSGs) also accepted additional BN heart grafts without further immunosuppression, though they rejected Piebald Virol Glaxo (PVG, RT1^c) rat heart grafts in the usual fashion. In the in vitro study, the proliferative response of the lymphocytes from LEW recipients with LSGs remained suppressed when they were stimulated by BN spleen cells, but not when stimulated by PVG cells. Bone marrow cells (BMCs) from LEW rats with LSGs showed strong, nonspecific, suppressive effects on the proliferative response in the mixed lymphocyte culture reaction, suggesting that one of the possible explanations for tolerance might be the involvement of a suppressor mechanism. Received: 7 August 1996 Received after revision: 12 February 1997 Accepted: 17 February 1997