输注Wnt3a基因修饰的骨髓间充质干细胞减轻小鼠急性移植物抗宿主病

目的 探讨联合输注经Wnt3a基因修饰的MSC减轻小鼠异基因骨髓移植(allo-BMT)后急性移植物抗宿主病(aGVHD)的作用及其可能机制.方法 以C57BL/6小鼠为供鼠,以Balb/c小鼠为受鼠,建立小鼠allo BMT模型.采用随机数字表法将受鼠分为4组:(1)移植对照组(A组):经受鼠尾静脉仅输注供鼠骨髓细胞5×106个;(2) aGVHD组(B组):经受鼠尾静脉输注供鼠脾细胞5×106个及骨髓细胞5×106个;(3)aGVHD+空载体组(C组):经受鼠尾静脉输注供鼠脾细胞5×106个、骨髓细胞5×106个及转染了空载体pAd-GFP的MSC 1×106个;(4)实验组(D组):经受鼠尾静脉输注供鼠脾细胞5×106个、骨髓细胞5×106个及经Wnt3a基因修饰的MSC1×106个.移植后监测各组受鼠的一般表现和存活情况,观察aGVHD的发生情况,检测受鼠脾脏中供者来源的T淋巴细胞数量变化及白细胞介素2(IL-2)和γ干扰素(IFN-γ)水平.结果 A组受鼠的存活时间均超过60d;B、C、D组受鼠的存活时间分别为(19.1±6.19)d、(32.6±19.6)d和(47.2±15.6)d,D组受鼠的存活时间较B组和C组明显延长(P＜0.05).移植后,B、C、D组受鼠的aGVHD评分分别为(8.0±0.41)分、(6.7±0.29)分和(4.0±1.0)分,D组受鼠的aGVHD评分较B组和C组明显降低(P＜0.05),且病理分级明显减轻.移植后3和5d时D组受鼠脾脏中供者T淋巴细胞的数量和增殖速度均较B组和C组明显降低(P＜0.05),并且移植后7、14、21、28 d时D组受鼠血清IL-2和IFN-γ水平均较B组和C组明显减少(P＜0.05).术后60d时,长期存活受鼠的骨髓细胞中H-2Kb细胞的嵌合率均在95％～100％.结论 联合输注经Wnt3a基因修饰的MSC可更有效的减轻小鼠allo-BMT后的aGVHD,这可能与Wnt3a的过表达激活了MSC的Wnt/β-catenin信号通路,从而抑制供者T淋巴细胞的早期激活和扩增及抑制IL-2和IFN-γ的表达有关.     

半相合骨髓移植术后并发急性移植物抗宿主病的护理

对26例半相合骨髓移植术后发生急性移植物抗宿主病(GVHD)病人进行治疗与护理,结果65.4％(17／26)GVHD病人的症状得到不同程度的控制或改善。提出做好皮肤、口腔、消化道、受损肝脏的护理及,心理护理,有利于病情改善。     

异体骨髓移植合并移植物抗宿主病的护理

近年来,骨髓移植(BMT)使得白血病、再生障碍性贫血的治愈机会明显增加,而移植物抗宿主病(GVHD)是异体骨髓移植(allo-BMT)的严重合并症之一,也是其主要死亡原因[1].因此,对GVHD的预防、观察及护理十分重要.我院1993年1月至1999年12月共施行25例allo-BMT,20例合并GVHD,临床护理如下.     

延迟并连续小鼠异基因骨髓移植减轻移植物抗宿主病的探讨

目的 探讨延迟并连续小鼠异基因骨髓移植减轻急性移植物抗宿主病(aGVHD)的作用及其机制.方法 选择BALB/c(H-2d)/b鼠为受者,C57BL/6(H-2b)小鼠为供者.受者接受60Coγ射线全身照射(TBI)后,建立骨髓移植模型.实验分为5组.对照组:TBI后4 h经受者尾静脉输注RPMI 1640液0.4 ml;经典移植组:TBI后4 h经受者尾静脉输注供者的骨髓细胞(BMC)和脾细胞(SC)各1×107个/0.2 ml;连续移植组:TBI后4 h、1 d、2 d和3 d分4次经受者尾静脉输注供者的BMC和SC,每次输注量各为2.5 × 106个/0.05 ml;延迟移植组:TBI后第4天经受者尾静脉输注供者的BMC和SC各1× 107个/0.2 ml;延迟并连续移植组:TBI后第4、5、6和7天分4次经受者尾静脉输注供者的BMC和SC,每次输注量各为2.5×106个/0.05 ml.移植后观察受者的临床表现、病理学改变及存活时间,采用酶联免疫吸附试验(ELISA)测定受者外周血中白细胞介素(IL)-2、IL-4、IL-10及γ干扰素(IFN-γ)的水平,应用流式细胞仪测定受者H-2b细胞、T淋巴细胞亚群及自然杀伤(NK)细胞的百分率.结果 对照组和经典移植组受者均在TBI后3周内因造血功能衰竭和/或aGVHD死亡;连续移植组和延迟移植组受者TBI后60 d存活率分别为30%和50%;延迟并连续移植组受者TBI后60 d的存活率为70%,高于其它4组(P＜0.05).延迟并连续移植组IL-4、IL-10的表达高于经典移植组(P＜0.05);IL-2和IFN-γ的表达低于经典移植组(P＜0.05).延迟并连续移植组TBI后20 d时外周血白细胞计数恢复正常,30 d时外周血T淋巴细胞亚群和NK细胞的表达恢复正常,60 d时H-2b细胞的百分率为(98.13±1.11)%.结论 延迟并连续小鼠异基因骨髓移植能减轻移植后的aGVHD.其作用机制主要是避开了TBI后炎症冈子表达的高峰期,下调了炎症因子和Th1类细胞因子的表达,促进了Th2类细胞因子的表达.     

间充质干细胞的免疫学特性及其在移植物抗宿主病中的作用

间充质干细胞是骨髓中发现的第二类干细胞,具有多向分化能力。本文就近年来间充质干细胞的免疫学特性研究进展及其在移植物抗宿主病等方面的临床应用情况作一综述。     

肝移植术后急性移植物抗宿主病

肝移植术后急性移植物抗宿主病是一种少见并发症,是来源于移植肝的同种反应性T细胞激活、增殖,达到一定数量对受体组织发起的破坏性的细胞免疫反应;主要临床表现为发热、皮疹、严重的腹泻和明显的低白细胞、低血小板血症,最终导致严重的感染和出血,但肝功正常.该病的诊断目前还缺乏敏感和特异性的方法,治疗主要依靠糖皮质激素、减低免疫抑制剂与支持治疗,但效果不佳,死亡率高达85%.     

巴利昔单抗在骨髓移植中防治急性移植物抗宿主病和免疫功能重建中的作用

目的 探讨抗CD25单克隆抗体（巴利昔单抗）在单倍体未去除T细胞的骨髓移植中防治重度急性移植物抗宿主病（aGVHD）的作用以及对免疫重建的影响。方法 1999年2月至2004年3月，对87例白血病患者进行了HLA单倍体未去T细胞的亲缘骨髓移植，其中2000年9月以前移植的15例受者预处理没有使用巴利昔单抗（对照组），2000年11月以后的72例预处理中加用巴利昔单抗（单抗组）。用流式细胞分析仪对受者移植术后1、3、6、9、12、18、24个月的外周血淋巴细胞亚群进行动态测定，对两组的结果进行比较。结果 移植后单抗组和对照组Ⅱ～Ⅳ度aGVHD的发生率分别为12．5％和33．3％（P=0.045）；与aGVHD相关的死亡率分别为6．9％和20％，两组差异有统计学意义。单抗组中9例临床诊断为Ⅱ～Ⅳ度aGVHD的患者因激素耐药再次使用巴利昔单抗治疗，缓解6例（66．7％），部分缓解2例，无效1例。移植后两组CD4^＋ T细胞的恢复均迟缓。单抗组CD4^＋ T细胞＞200个／μ1的时间为移植后6个月，在移植后18个月绝对数恢复正常。CD3^＋、CD8^＋和CDl9^＋ B细胞在9～12个月恢复正常水平，两组淋巴细胞亚群的恢复情况差异无统计学意义。结论 使用抗CD25单克隆抗体预防和治疗受者严重的aGVHD有一定疗效；受者白血病的复发率和移植后的感染率无明显增加；对骨髓移植后淋巴细胞亚群数量的重建无明显的影响。     

脐带间充质干细胞治疗难治性慢性移植物抗宿主病的疗效观察

目的 探讨脐带来源间充质干细胞(MSC)治疗异基因造血干细胞移植术后难治性慢性移植物抗宿主病(cGVHD)的疗效及安全性。 方法 7例恶性血液病患者接受异基因造血干细胞移植术后出现cGVHD,常规免疫抑制剂治疗无效,在原有免疫抑制剂基础上给予脐带来源MSC治疗,细胞数1×10⁶/kg,每周1次,共4次。观察其疗效、安全性和患者存活情况。 结果 7例患者接受MSC输注后,2例获得完全缓解(CR),3例部分缓解(PR),总有效5例(5/7),2例无效(NR)。未发现输注相关不良反应,无患者出现原发病复发。1例患者部分缓解后继发巨细胞病毒肺炎,死于重症肺炎,其余病例均存活。 结论 脐带MSC对于治疗难治性cGVHD具有一定的疗效,且输注过程安全。      

肝移植术后急性移植物抗宿主病

肝移植术后急性移植物抗宿主病是一种少见并发症,是来源于移植肝的同种反应性T细胞激活、增殖,达到一定数量对受体组织发起的破坏性的细胞免疫反应;主要临床表现为发热、皮疹、严重的腹泻和明显的低白细胞、低血小板血症,最终导致严重的感染和出血,但肝功正常。该病的诊断目前还缺乏敏感和特异性的方法,治疗主要依靠糖皮质激素、减低免疫抑制剂与支持治疗,但效果不佳,死亡率高达85%。     

脐带间充质干细胞治疗移植物抗宿主病患者的护理

目的总结18例异基因造血干细胞移植术后移植物抗宿主病患者进行脐带间充质干细胞治疗的护理经验。方法对18例异基因造血干细胞移植术后移植物抗宿主病患者进行脐带间充质干细胞治疗,脐带间充质干细胞细胞数量为107/U,患者每次均输入5U。结果 18例患者输注脐带间充质干细胞后+30d进行追踪观察疗效评价,完全缓解8例,部分缓解8例,无效2例(因Ⅳ度移植物抗宿主病合并肺部感染死亡)。结论细致的病情观察及执行规范的脐带间充质干细胞输注,是治疗异基因造血干细胞移植移植物抗宿主病患者的护理重点。     

Ly49A转基因淋巴细胞对异基因骨髓移植后GVHD和GVL的影响

目的　观察Ly49A基因转染淋巴细胞对异基因骨髓移植后移植物抗宿主病 (GVHD)和移植物抗白血病效应 (GVL)的影响。方法　经逆转录病毒介导将Ly49A基因转染至C57BL/ 6小鼠的淋巴细胞 ,以C57BL/ 6(H 2 b)小鼠为供者 ,以接种EL961 1红白血病细胞的BALB/c(H 2 d)小鼠为受者 ,进行脾淋巴细胞和骨髓细胞联合移植 ,建立异基因急性GVHD模型 ,观察Ly49A基因转染的淋巴细胞对GVHD和GVL的影响。结果　在未进行移植的单纯照射组 ,小鼠的存活时间为 (6 .50±2 .41 )d ;仅以环磷酰胺治疗的小鼠存活时间为 (2 0 .90± 2 .88)d ;非转染淋巴细胞和骨髓细胞联合移植组的存活时间为 (1 7.1 0± 4 .65)d ;空载体转染淋巴细胞和骨髓细胞联合移植组的存活时间为(1 7.40± 5 .32 )d ;Ly49A转染淋巴细胞与骨髓细胞联合移植组的存活时间为 (35 .2 0± 1 2 .52 )d ,较上述各组明显延长 (P =0 .0 0 0 )。结论　Ly49A基因转染的淋巴细胞在异基因骨髓移植模型上具有一定程度的降低GVHD并保留GVL的作用     

Acute bleeding after allogeneic bone marrow transplantation: association with graft versus host disease and effect on survival

BACKGROUND: Hemorrhagic complications are frequently implicated clinically for the high morbidity and mortality of acute graft versus host disease (GVHD), however, only few reports characterize the incidence and timing of bleeding in relation to GVHD, and essentially no study has quantified the effect of bleeding on survival of allogeneic patients with GVHD. This study examines the association of bleeding with acute GVHD and the effect of both complications on survival. METHODS: A total of 463 allogeneic patients transplanted at the Johns Hopkins Hospital, were included in the study. Bleeding evaluation was based on daily scores of intensity and blood transfusions. All bleeding sites were recorded. GVHD staging was defined by the extent of rash, serum bilirubin, diarrhea, and confirmatory histology. RESULTS: The incidence of GVHD was 27.4%, bleeding occurred in 40.2%. The incidence of bleeding was higher in patients with GVHD as compared with non-GVHD, and correlated with GVHD severity. The higher bleeding incidence in GVHD was due to gastrointestinal hemorrhage, hemorrhagic cystitis, and pulmonary hemorrhage. While the majority of bleeding (51/75) in non-GVHD patients initiated within 30 days after bone marrow transplantation (BMT), only 32.3% (21/65) of the bleeding in the GVHD group initiated within 30 days, and the risk for bleeding continued until day 100. Bleeding was a late event compared to GVHD, however, most bleeding episodes were associated with active GVHD. Both GVHD and bleeding were individually associated with reduced survival, with profound additive adverse effect: median survival in 221 nonbleeding non-GVHD was >83.2 months, GVHD nonbleeding (39 patients) had median of 10.6 months, bleeding non-GVHD (99 patients) had median of 4.3 months, and median survival of the GVHD bleeding group (85 patients) was 3.2 months. CONCLUSIONS: Our results support an association of bleeding with acute GVHD, suggesting that GVHD is a risk factor for bleeding after BMT. The occurrence of bleeding clearly identified poor outcome subgroup within GVHD, suggesting further evaluation for clinical application of bleeding in the assessment of GVHD severity.     

Airways obstruction associated with graft versus host disease after bone marrow transplantation.

Eight patients, five with chronic granulocytic leukaemia and three with severe aplastic anaemia, developed moderately severe airflow obstruction after allogeneic bone marrow transplantation. All eight had clinically and radiologically normal lungs before undergoing transplantation. Treatment in the patients with chronic granulocytic leukaemia before transplantation included high dose total body irradiation. All eight patients developed acute and chronic graft versus host disease after transplantation. The pulmonary syndrome consisted of cough, dyspnoea, and wheezing beginning six to 20 weeks after transplantation, with ratios of forced expiratory volume in one second (FEV1) to vital capacity (VC) falling to 60% or less of predicted values. The three patients with severe aplastic anaemia had relatively mild graft versus host disease and acute chest infection may have initiated or contributed to their airways obstruction, which subsequently resolved. The five patients with chronic granulocytic leukaemia had more severe graft versus host disease and more progressive respiratory problems; two died and three continued to have persistent airflow obstruction 11, 15, and 20 months after transplantation. None of those with chronic granulocytic leukaemia improved. Transfer factor (TLCO) was reduced in all patients after bone marrow transfer and did not improve; in the patients with chronic granulocytic leukaemia the reduction in TLCO preceded the fall in FEV1/VC ratio. Open lung biopsy in one of the patients with chronic granulocytic leukaemia showed obliterative bronchiolitis with lymphocytic infiltration consistent with graft versus host disease. Bronchodilators were of no benefit in the management of these patients, but prompt treatment of infection and early use of corticosteroids may have contributed to the improvement seen in the patients with severe aplastic anaemia.     

The effect of peripheral lymphoid cells on the incidence of lethal graft versus host disease following allogeneic mouse bone marrow transplantation

Experiments were performed to study the role of circulating lymphoid cells in the incidence of lethal graft versus host disease (GVHD) in radiation-induced fully allogeneic mouse chimeras. The incidence of GVHD was reduced significantly in BALB/c → C57BL/6 radiation chimeras if bone marrow donors were exsanguinated immediately prior to marrow harvest. Chimeras resulting from the injection of bone marrow from bled donors exhibited only donor cells in spleen, bone marrow and peripheral blood and normal levels of Thy 1+ and Ia+ cells were found in each of these lymphoid compartments. The addition of as few as 3 × 10⁴ peripheral mononuclear cells to the marrow from exsanguinated donors uniformly led to lethal GVHD. ⁵¹Cr-labeled cell traffic studies revealed that prior exsanguination of marrow donors led to about a 70% reduction in the number of circulating mononuclear cells contaminating the bone marrow at the time of marrow harvest. This decrease in contaminating peripheral cells was calculated to be in the appropriate range to account for the decreased GVHD seen when marrow from exsanguinated donors was used. It thus appears that peripheral cells contaminating marrow can be an important factor in causing lethal GVHD in allogeneic radiation chimeras. These results raise the possibility that the fulminant GVHD seen in human marrow transplantation is in part due to the major contamination of bone marrow with peripheral blood that results from the techniques currently used for human bone marrow harvest.     

Prediction of graft versus host disease by frequency analysis of cytotoxic T cells after unrelated donor bone marrow transplantation

HLA "matched" unrelated donor bone marrow transplants are associated with an increased incidence and severity of graft-versus-host disease in comparison with HLA-identical sibling transplants. This is presumably due to HLA and non-HLA histocompatibility differences between donor and recipient. Using a limiting dilution assay, we have previously demonstrated a relationship between cytotoxic T lymphocyte precursor frequency and HLA disparity. In this study we have compared CTL-p frequencies with clinical GVHD, and demonstrate for the first time a significant correlation (P less than 0.005) between high CTL precursor frequency prior to BMT and severity of acute GVHD after HLA A, B, DR "matched" unrelated donor transplants using T cell depleted marrow. This assay system may be of value in the final selection of HLA "matched" unrelated donors for BMT.     

Obstructive lung disease after allogeneic bone marrow transplantation

We report the cases of 3 patients with marked dyspnea and an obstructive ventilation disorder associated with chronic graft-versus-host disease after allogeneic bone marrow transplantation. This disorder was characterized by recurrent pulmonary infections and colonization of the lower respiratory tract by Pseudomonas aeruginosa. Two patients have shown rapidly progressive deterioration with death following due to respiratory failure. Intensive therapy with antibiotics, bronchodilators, high-dose steroids, and azathioprine was not effective in arresting the malignant course of this disorder.     

第三方脐血预防异基因造血干细胞移植后移植物抗宿主病

目的 探讨无关供者和单倍型亲缘供者异基因造血干细胞移植(allo-HSCT)前输注第三方脐血预防移植物抗宿主病(GVHD)的效果.方法 2007年至2011年接受无关供者及单倍型亲缘供者allo-HSCT的受者共41例.脐血预防组(25例),于移植前1d予以HLA配型为4/6～6/6的第三方脐血细胞输注,平均输入有核细胞数为(1.64±0.49)×107/kg,次日再输入供者造血干细胞.其余患者作为对照组(16例).两组均采用抗胸腺细胞球蛋白+环孢素A+甲氨蝶呤+吗替麦考酚酯预防急性GVHD.比较两组间急性GVHD的发生率、严重程度以及移植相关死亡率.结果 脐血预防组和对照组aGVHD累积发生率分别为44.0％(11/25)和68.8％(11/16),两组比较,差异无统计学意义(x2=2.403,P＞0.05);Ⅲ～Ⅳ度aGVHD累积发生率分别为16.0％(4/25)和37.5％(6/16),两组比较,差异无统计学意义(x2=2.445,P＞0.05),100 d治疗相关病死率分别为12.0％(3/25)和12.5％(2/16),两组比较,差异无统计学意义(x2=0.002,P＞0.05).结论 在无关供者及单倍型亲缘供者allo-HSCT前应用第三方脐血细胞,可能有效预防GVHD的发生或减轻GVHD的严重度,但尚需要进一步设计扩大规模的随机对照临床试验验证其有效性.     

利用脊柱骨来源骨髓细胞建立急性移植物抗宿主病模型

目的探讨利用脊柱骨来源骨髓细胞建立小鼠异基因造血干细胞移植(allogeneic hematopoietic stem cell transplantation,Allo-HSCT)急性移植物抗宿主病(aGVHD)模型的可行性。方法选择C57BL/6(H-2b)雄性小鼠为供体鼠,BALB/c(H-2d)雌性小鼠为受体鼠。制备供体鼠的脾细胞和脊柱骨来源骨髓细胞悬液。受体鼠采用药物加小剂量辐照的预处理方式,于移植前8d～移植前4d腹腔注射氟达拉滨(200mg/kg),接着移植前3d～移植前1d腹腔注射环磷酰胺(60mg/kg),最后在移植前进行全身照射(total-body irradiation,TBI),照射剂量为4Gy(戈瑞)。18只受体鼠经预处理后随机分为3组,每组6只:(1)骨髓移植组,只输入1×107个脊柱骨来源骨髓细胞;(2)aGVHD组,输注1×107个脊柱骨来源骨髓细胞和5×106个脾细胞,建立aGVHD模型;(3)空白对照组,不输入任何细胞。观察3组小鼠生存状态及存活率,取aGVHD组与骨髓移植组存活21d的受体鼠进行病理学检查,取aGVHD组移植后21～28d存活的小鼠的脾脏进行流式细胞术检测骨髓细胞嵌合度。结果骨髓移植组小鼠全部存活,可重建造血,单纯输注骨髓细胞不会诱发aGVHD。aGVHD组小鼠出现aGVHD表现,100%发生aGVHD相关死亡,中位生存期为18d;病理检查结果显示符合aGVHD病理表现,移植后21～28d存活的小鼠诊断为供受体混合嵌合状态,符合aGVHD诊断标准。结论用脊柱骨来源骨髓建立的aGVHD模型完全符合标准,且更加经济,适合大规模建模。     

骨髓间充质干细胞促进大鼠小体积肝移植后肝再生的实验研究

目的 探讨骨髓间充质干细胞能否促进大鼠小体积肝移植后移植肝的再生.方法 体外分离、培养、鉴定大鼠骨髓间充质干细胞.在30％大鼠部分肝移植的模型基础上,实验分为对照组(30％ PLT)和骨髓间充质干细胞干预组(30％PLT+ MSC).比较两组肝移植术后的存活率,分析肝功能的变化,通过免疫组化来观察移植肝标本Cyclin D1和PCNA的表达,并对移植肝组织结构进行电镜观察.结果 骨髓间充质干细胞的干预,能够改善小体积肝移植术后肝功能状况,减轻组织学损伤,并能够提高存活率,30％ PLT组与30％ PLT+ MSC组一周存活率分别为16.7％和58.3％.而在Cyclin D1和PCNA的免疫组化表达中,30％ PLT组表达明显抑制,30％ PLT+ MSC组表达上调.结论 骨髓间充质干细胞可以存进大鼠小体积肝移植后移植肝的再生,改善肝功能,并提高存活率.     

骨髓间充质干细胞移植对去卵巢骨质疏松骨量影响的实验研究

目的 探索骨髓间充质干细胞（BMSCs）移植对去卵巢骨质疏松大鼠骨密度的影响。方法 雌性SD大鼠随机分为空白对照组（A组）、模型组（B组）、细胞治疗组（C组）、雷诺昔芬药物治疗组（D组）。通过去卵巢建模成功后,C组通过尾静脉移植BMSCs,D组口服雷诺昔芬抗骨质疏松药物。结果 与A组相比,B组腰椎和股骨骨密度均明显降低（P＜0.01）。MSCs治疗后,C 组中的骨密度得到明显改善,并与B组比较具有统计学差异（P＜0.01）,亦优于D组。结论 BMSCs可以有效改善去卵巢大鼠骨质,这为绝经后骨质疏松的治疗提供了一种新的方法,为进一步临床应用提供了实验支持。