干细胞因子受体基因在再生障碍性贫血患儿中的表达

目的探讨再生障碍性贫血（再障）患儿造血干细胞因子受体（C-KIT）基因Exon9、11、13的表达和序列构象及与发病机制的关系。方法应用逆转录-聚合酶链反应（RT-PCR）和聚合酶链反应单链构象多态分析（PCR-SSCP）和序列测定技术,测定再障与正常对照儿童各15例C-KIT基因Exon9、11、13的表达水平和结构是否存在变异。结果1.再障组C-KIT基因Exon9、11、13阳性表达率（40.0%）与正常对照组（46.67%）比较无显著性差异（P〉0.05）;2.经PCR-SSCP分析和基因序列测定C-KIT基因Exon9、11、13均未见碱基序列改变或碱基数目增多、缺失。结论C-KIT基因Exon9、11、13表达与儿童再障发病关系不大,提示儿童再障的发病可能不是由于Exon9、11、13突变引起。     

慢性再生障碍性贫血患儿血浆可溶性干细胞因子及其受体水平测定的意义

目的探讨可溶性干细胞因子(SCF)及其受体(s-kit)与儿童慢性再生障碍性贫血(CAA)发病的关系。方法利用ELISA法测定CAA患儿、正常儿童外周血浆及脐血血浆s-kit及SCF水平。结果CAA组血浆s-kit水平(13.02±5.18)μg/L低于正常儿童组(17.82±5.36)μg/L和脐血组(19.0±5.63)μg/L,CAA组血浆SCF水平(365.5±98.35)μg/L,低于正常儿童组(469.26±102.32)μg/L和脐血组(567.22±135.88)μg/L。结论CAA组血浆s-kit及SCF水平降低,提示s-kit及SCF可能在儿童CAA发病中起一定作用。     

Efficacy and Safety of Human Umbilical Cord Derived Mesenchymal Stem Cell Therapy in Children with Severe Aplastic Anemia Following Allogeneic Hematopoietic Stem Cell Transplantation: A Retrospective Case Series of 37 Patients

The treatment of pediatric severe aplastic anemia (SAA) with allogeneic hematopoietic stem cell transplantation (allo-HSCT), presents major challenges including the risks of graft failure, septic complications, and graft-versus-host disease (GVHD). Additive infusions of human umbilical cord derived mesenchymal stem cell (hUC-MSC) may be administered to improve patient survival. We retrospectively examined 37 pediatric patients with SAA who received allo-HSCT and subsequent infusions of hUC-MSC suspension at a dose of 1.0 × 10⁶ /kg. The times and doses of hUC-MSC infusions were increased in patients with severe GVHD. All patients received hUC-MSC infusions. The median time to post-transplantation neutrophil count of greater than 0.5 × 10⁹ /L was 14 days (range, 11–20 days) and time to post-transplantation platelet count of greater than 20 × 10⁹ /L was 19 days (14–29 days). The overall frequency of acute GVHD (aGVHD) was 45.9% (17/37). These aGVHD episodes occurred at a median time of post-transplantation 47 days (15–83 days). The frequency of chronic GVHD (cGVHD) was 18.9% (7/37); cGVHD developed from aGVHD in 10.8% (4/37) of patients. The GVHD-associated mortality rate was 18.9% (7/37) and aGVHD-specific mortality rate was 8.1% (3/37). The median overall survival time was 35 months (9—67 months) and the three-year overall survival rate was 74.2% (28/37). Seven patients died of GVHD, one patient died of a severe invasive fungal infection, and one patient died of renal failure. In conclusion, post-transplantation hUC-MSC infusions seemed to be safely infused in children with SAA who have previously received allo-HSCT.     

异基因造血干细胞移植治疗儿童重型再生障碍性贫血

目的探讨异基因造血干细胞移植(allo-HSCT)治疗儿童重型再生障碍性贫血(SAA)的疗效及并发症。方法 4例SAA患儿,均接受氟达拉滨、环磷酰胺、抗胸腺细胞球蛋白预处理;其中3例患儿行HLA全相合同胞骨髓造血干细胞移植(BMT),1例患儿行HLA全相合同胞外周血造血干细胞移植(PBSCT)。同胞供者采集重组人粒细胞集落刺激因子5μg.kg-1.d-1,动员骨髓及外周血干细胞。采用环孢素+短疗程小剂量甲氨蝶呤方案预防移植物抗宿主病,前列腺素E预防肝静脉闭塞综合征,更昔洛韦预防巨细胞病毒感染,美司那及水化碱化预防出血性膀胱炎。通过DNA短串联重复序列多态性分析检测植入情况。结果 2例BMT患儿及1例PBSCT患儿完全植入;1例BMT患儿嵌合植入。中性粒细胞>0.5×109L-1中位时间12 d(9～15 d),血小板>20×109L-1中位时间19 d(12～30 d)。结论 allo-HSCT是治疗儿童SAA的有效方法,维持造血功能以及移植后并发症的发生及防治,仍是目前重点讨论的课题。     

再生障碍性贫血患儿骨髓内人巨细胞病毒活动性感染对造血功能的影响

目的探讨再生障碍性贫血(再障)患儿骨髓内人巨细胞病毒(HCMV)活动性感染与骨髓CD34 细胞/有核细胞的百分比之间的关系,了解骨髓HCMV活动性感染对再障患儿骨髓造血功能的影响。方法收集23例再障初诊患儿(再障组),5例非血液病患儿作为对照。所有患儿均于治疗前抽取骨髓液标本,一部分骨髓液采用Trizol法抽提骨髓液RNA,然后进行反转录聚合酶链反应,扩增后通过琼脂糖凝胶电泳检测患儿骨髓内是否存在HCMV活动性感染;将再障组和对照组患儿另一部分骨髓液进行多参数流式细胞术检测,分别检测二组患儿骨髓CD34 细胞/有核细胞百分比。结果23例再障患儿中9例患儿骨髓标本内可检测到HCMV RNA表达,阳性率为39.1%;5例对照组患儿骨髓标本内均未检测到HCMV RNA表达。流式细胞术检测显示再障患儿骨髓CD34 细胞/有核细胞为(0.27±0.21)%,明显低于对照组[(1.57±0.45)%](P<0.05)。骨髓HCMV RNA阳性再障患儿的CD34 细胞/有核细胞为(0.13±0.07)%,显著低于HCMV RNA阴性患儿[(0.36±0.22)%](P<0.05)。结论HCMV可感染再障患儿的骨髓组织,其活动性感染可导致骨髓CD34 细胞/有核细胞的百分比明显减少,提示骨髓内HCMV活动性感染对骨髓造血功能有抑制作用。     

Cyclosporine a in Five Children with Severe Aplastic Anemia

Five children with aplastic anemia (AA) were treated with Cyclosporine A (CyA) after failure or poor response to other immunosuppressive agents. A complete remission was achieved in 3 patients, a good partial response in 1, and a poor partial response in another. In 1 patient in whom a complete remission was obtained, a relapse occurred 4 months after CyA was stopped. The treatment was resumed and a complete remission was again observed. Four patients were still on treatment, and side effects were mild. CyA appeared to be an effective treatment in AA.     

再生障碍性贫血患儿骨髓祖细胞培养的临床意义

目的探讨再生障碍性贫血(AA)患儿红细胞集落生成单位(CFU-E)、爆式红细胞集落生成单位(BFU-E)、粒细胞-单核细胞集落生成单位(CFU-GM)及巨核细胞集落生成单位(CFU-Meg)骨髓祖细胞培养情况,了解骨髓祖细胞在AA发病中的作用及意义。方法抽取28例AA患儿骨髓3～4mL,以淋巴细胞分离液提取单个核细胞,用磷酸盐缓冲液(PBS)洗涤2次,调节单个核细胞水平为106/mL,取0.3mL分别接种于红系、粒-单核系、巨核系培养基分别作4类祖细胞培养,在7、14d测定其集落数。结果28例AA中,4类集落数均值与对照组比较,差异有显著意义(P<0.01或P<0.001)。集落数随病情加重而逐步下降。4类集落数低于对照组患儿下限所占百分比分别为BFU-E35.71%、CFU-E85.71%、CFU-GM75.00%、CFU-Meg89.29%。治愈及进步者4类集落数接近对照组水平,治疗无效及进展者与治疗前比较无变化。结论动态监测AA患儿骨髓4类祖细胞集落数变化对辅助诊断、观察疗效及预后判断均有指导意义。     

再生障碍性贫血患儿外周血淋巴细胞表型分析

目的　探讨再生障碍性贫血 (再障 )患儿外周血淋巴细胞表型的变化 ,了解其在发病中的作用及临床意义。方法　将 1 1 5名确诊为再障的患儿取外周血作淋巴细胞表型分析并与正常组比较。结果　再障组CD4 % ,CD8 % ,CD4/CD8分别为 2 9.53± 8.94,34 .2 5± 1 0 .38,0 .96± 0 .53 ,对照组为 35 .2 5± 5 .1 6 ,2 5 .0 8± 4 .34 ,1 .48± 0 .46 ,两者比较有显著差异 (P <0 .0 0 5) ,而重型再障的细胞表型CD4/CD8、γδT细胞百分率、HLA DR %则更高于慢性再障 ,两者有显著差异。CD3 %两组分别为 69.53± 2 4 .35和 69.98± 5 .79,两者比较无显著差异 (P <0 .0 5)。结论　再障患儿存在严重的免疫异常 ,且与疾病严重程度呈正相关 ,临床可行免疫抑制治疗。     

SINGLE-CENTER EXPERIENCE: Immunosuppressive Therapy as Frontline Treatment for 33 Children with Acquired Severe Aplastic Anemia

The authors retrospectively analyzed the records of 33 children with acquired severe aplastic anemia (SAA) diagnosed from July 1998 to October 2007 and first treated by immunosuppressive therapy (IST). Serial hematologic parameters, complications, transfusion requirements, and time to response were assessed. Allogeneic hematopoietic stem cell transplantation (HSCT) was attempted in 7 patients after failure of IST (n = 6) or relapse following an initial response to IST (n = 1). One child died of post-transplant lymphoproliferative disorder. Thirty of the 33 patients are alive and well after a median follow-up of 45 months (range, 7–116 months). Overall (transfusion-independent) response to IST was 73% (24/33). The actuarial 5 years survival rate was 89.4%. In this study, all patients with SAA received IST as standard front-line therapy. Approximately three-fourths of patients with SAA have durable recovery and excellent overall survival.     

Successful Bone Marrow Transplant and Re-Growth of Hair in a Patient with Posthepatic Aplastic Anemia Complicated by Alopecia Totalis

Bone marrow transplant was performed in a child with posthepatic aplastic anemia complicated by alopecia totalis. The procedure was successful, as the graft was apparently "taken" by the recipient and hair started to grow on the head. The hair has not been lost since, and is still growing as of the date of this report. Although nonA nonB hepatitis virus was found to be the cause of hepatitis in this patient, the reason for the alopecia totalis which developed 7 years previously is unclear. The postoperative growth of the hair was attributed to restoration of normal immunologic function through bone marrow transplant.     

骨髓间充质干细胞在先天性脊椎裂胎鼠脊髓中的分化

目的观察大鼠骨髓间充质干细胞(MSC)在先天性脊椎裂胎鼠脊髓中的存活与分化,探寻细胞替代治疗先天性脊椎裂的方法。方法用贴壁培养法进行大鼠MSC原代培养并传代,携带增强型绿色荧光蛋白(EGFP)基因的腺病毒转染第3代骨髓MSC。将转染的MSC注射到先天性脊椎裂胎鼠的脊髓中。母鼠孕20 d时取先天性脊椎裂胎鼠的脊椎,固定脱水后做冷冻切片,免疫荧光方法检测脊髓中EGFP阳性MSC中巢蛋白(Nestin)、胶质纤维酸性蛋白(GFAP)的表达。结果骨髓MSC中CD90阳性细胞占99%,CD44阳性细胞占95%,不表达CD34。腺病毒-EGFP转染骨髓MSC的转染率为61%。致畸组显性脊椎裂胎鼠占64.3%。移植的MSC存在于脊髓表面或进入脊髓中,部分细胞变为长梭形或多角形。免疫荧光法检测结果显示移植到脊髓中的MSC可表达神经干细胞的标志物Nestin和神经胶质细胞的标志物GFAP。结论带有EGFP的MSC经细胞移植后能在胎鼠脊髓内存活,并能分化为神经干细胞和神经胶质细胞。     

当归注射液对免疫诱导再生障碍性贫血小鼠骨髓细胞组织形态及超微结构的影响

目的　探讨当归注射液对免疫诱导再生障碍性贫血 (AA)小鼠骨髓细胞组织形态及超微结构的影响。方法　雌性Balb/c小鼠 ,随机分为正常组、模型组、治疗组和对照组。于d1 2每只小鼠采集尾静脉血测外周血白细胞计数 ,后断颈处死小鼠 ,取出尺骨及股骨 ,计数骨髓单个核细胞。并行尺骨组织学切片及超微结构观察。结果　与正常组比较 ,模型组造血细胞明显减少 ,骨髓微环境受损 ,治疗组能明显改善骨髓增生程度 ,修复骨髓微环境 ,增加骨髓单个核细胞计数 ;与对照组比较无显著差异。结论　当归注射液能通过促进骨髓造血细胞增生 ,改善骨髓微环境对免疫诱导AA小鼠进行保护作用。     

胶质细胞源性神经营养因子和神经营养素-3双基因转染大鼠骨髓间充质干细胞体外诱导分化为神经样细胞的研究

目的 探讨胶质细胞源性神经营养因子(GDNF)和神经营养素3(NT-3)双基因修饰诱导的大鼠骨髓间充质干细胞(BMSCs)分化为神经样细胞的可行性以及GDNF和NT-3的表达变化.方法 全骨髓法分离培养BMSCs,流式细胞术检测BMSCs标志CD90和造血干细胞标志CD45.转染带荧光的GDNF和NT-3基因,在荧光显微镜下观察绿色荧光蛋白(GFP)的表达及细胞形态变化;免疫荧光检测神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF)和神经胶质酸性蛋白(GFAP)表达;Western blot 检测细胞中GDNF及NT-3蛋白表达.对照组为未转染GDNF和NT-3基因的BMSCs.结果 BMSCs能在体外成功分离培养,细胞高表达CD90(92.7%),不表达CD45.诱导分化后,BMSCs胞体变圆,伸出明显突起,并可见多数细胞相互交织成网状结构,呈神经细胞样形态.免疫荧光标记检测可见实验组细胞表达NSE和NF,而不表达GFAP.而对照组阴性.Western blot检测可见细胞GDNF及NT-3蛋白表达增强.结论 GDNF和NT-3双基因修饰诱导的BMSCs可分化为神经样细胞并表达神经元的标志,为基因治疗神经系统疾病如先天性巨结肠提供实验基础.     

Effect of Recombinant Human Granulocyte-Colony Stimulation Factor (rhG-CSF) on Immune System in Pediatric Patients with Aplastic Anemia

To determine the effect of recombinant human granulocyte-colony stimulation factor (rhG-CSF) on the immune system, serum immunoglobulins, lymphocyte subsets, and serum cytokines were analyzed in eight pediatric patients with atlastic anemia (AA) during 8-week rhG-CSF therapy. The rhG-CSF was administered either subcutaneously (200 μglm^' × 4 weeks, followed by 400 Fglm' × 4 weeks) or intravenously (400 μglm^' × 4 weeks, followed by 800 μg/m² × 4 weeks). In response to rhG-CSF therapy, neutrophil counts exceeded the pretreatment counts by twofold during the first week except for one case that did not attain twofold increase until day 41. While serum IgC and IgA were not affected, serum IgM was elevated during treatment in six of the eight cases to more than 1.2-fold basal levels (P < 0.04); however, there was no increase in serum interleukin (IL)-6 and interferon-y levels. On the other hand, CD56 positive NK cells significantly dropped from 7.7% to 4.5% (P < 0.02). These results indicate that systemic administration of rhG-CSF affects not only the neutrophil count, but also serum IgM levels and the natural killer cell population in patients with AA.     

儿童骨髓间充质干细胞体外培养与富集效率的研究

目的比较密度梯度离心法和全骨髓培养法分离培养儿童骨髓间充质干细胞(BMSCs)获得的细胞纯度与克隆形成效率。方法取等量抗凝的儿童骨髓分别以密度梯度离心法和全骨髓培养法分离培养BMSCs,传代至第3代分别计数2种方法所得BMSCs的细胞总数,流式细胞仪检测表面标记物及其纯度,对第5代BMSCs分别绘制生长曲线,全骨髓培养法培养至第6代流式细胞仪再检测表面标记物及其纯度。结果2种方法均能有效培养出BMSCs,BMSCs强表达CD105及CD13,低表达CD34;3mL骨髓样本用全骨髓培养法扩增至第3代平均可获得3.15×107个细胞,而密度梯度离心法3mL骨髓只获得1.08×107个细胞,二者有显著性差异(P<0.05);密度梯度离心法第3代BMSCs纯度达95%以上,全骨髓培养法第3代BMSCs纯度约85%,多次换液传至第6代纯度可达到95%以上;2种方法第5代BMSCs生长曲线近似,接种后0～3d为生长潜伏期,3～6d为对数生长期,之后进入平台期,无显著性差异(P>0.05)。结论密度梯度离心法分离培养BMSCs,可在短时间内获取纯度较高的BMSCs;全骨髓培养法分离培养BMSCs,可从少量标本获得最大数量的BMSCs,多次换液传代也可获得纯度较高的BMSCs。     

抗淋巴细胞球蛋白治疗再生障碍性贫血的疗效及不良反应

目的 探讨抗淋巴细胞球蛋白(ALG)治疗再生障碍性贫血(AA)的疗效.分析ALG不良反应的特点和防治方法.方法对2002年4月-2009年10月本院收治的162例诊断为AA并接受ALG治疗的患儿的临床资料进行回顾性分析,共154例完成了ALG 疗程.其中极重型AA(VSAA)34例,重型AAⅠ型(SAAⅠ)103例,重型AAⅡ型(SAAⅡ)7例,中型AA(MAA)10 例.结果 154例中,基本治愈62例(40.3%),缓解15例(9.7%),进步28例(18.2%),无效或死亡23例(14.9%),失访26例(16.9%),总有效率为68.2%,复发8例(5.2%).63例出现变态反应,主要表现为发热和皮疹等.发生时间为用药1～5 d,持续1～4 d.8例因严重变态反应而停止使用ALG,其中1例死于严重的全身血管性变态反应.81例出现血清病反应,主要表现为发热、皮疹和关节肿痛等.发生时间为ALG治疗结束2～14 d,持续1～12 d.有无血清病与ALG疗效差异无统计学意义(P>0.05).患儿性别、年龄、CD4/CD8、人白细胞Dr抗原与血清病发生率的差异均无统计学意义(Pa>0.05).结论 ALG治疗AA疗效肯定,变态反应和血清病为治疗中常见的不良反应,应用甲泼尼龙可较好地控制过敏和血清病症状.     

体外诱导大鼠骨髓间质干细胞分化为胰岛素分泌细胞(英文)

目的：探索大鼠骨髓间质干细胞体外诱导分化为胰岛素分泌细胞的方法。为解决胰岛移植物来源匮乏这一问题提供新的思路。方法：采用横向分化技术,将成年大鼠骨髓间质干细胞诱导成为胰岛素分泌细胞。间接免疫荧光法鉴定诱导前后细胞nestin、胰岛素、胰高血糖素、生长抑素及Pdx-1的表达,RT-PCR法检测诱导前后细胞nestin,胰岛素-1,葡萄糖转运子-2,葡萄糖激酶及其转录因子Isl-1,Pdx-1,Pax-4和Pax-6 mRNA的表达;测定24 h胰岛素分泌量和胰岛素刺激实验评价诱导前后细胞的功能。结果：诱导5 h,nestin阳性细胞为(44.6±7.3)％;诱导24 h,nestin阳性细胞增至(61.8±8.4)％;此后,nestin阳性细胞数目开始下降,诱导第14天后,nestin表达基本消失。诱导后的胰岛素分泌细胞可以表达胰岛素、胰高血糖素、生长抑素和Pdx-1等蛋白;表达胰岛素-1、葡萄糖转运子-2、葡萄糖激酶及其多种转录因子mRNA;胰岛素分泌量增加;胰岛素刺激实验反应敏感等。而诱导前MSCc不县备上述特点。结论：大鼠骨髓间质干细胞体外可以诱导成为胰岛素分泌细胞,为胰岛移植开辟新的研究思路。     

骨髓间充质干细胞移植对肺动脉高压大鼠肺血管重构的影响

目的 探讨骨髓间充质干细胞(MSCs)移植对野百合碱诱导的肺动脉高压(PAH)大鼠肺血管重构的影响.方法 体外分离、培养并纯化SD大鼠骨髓MSCs.健康雄性SD大鼠随机分为3组:正常对照组(n＝15)、PAH组(n＝20)和MSCs移植组(n＝20).PAH组和MSCs移植组大鼠一次性项背部皮下注射野百合碱(50 mg·kg -1),复制PAH肺血管重建动物模型.3组大鼠在同等条件下饲养.3周后,MSCs移植组大鼠经舌下静脉注射5×109 L-1的经Hoechst 33342标记的MSCs细胞悬液1 mL,PAH组大鼠予等量低糖-Dulbecco改良Eagle's培养液(L-DMEM)注射,正常对照组不做任何处理.移植28 d,观察3组大鼠肺小动脉的显微结构及超微结构的改变.结果 PAH大鼠用MSCs移植28 d后,肺小动脉管壁厚度指标(WD/TD％、VA％)、放射性肺泡计数、肺非肌性小动脉肌化程度均较PAH组显著改善(P＜0.01);其肺小动脉的重构及超微结构的血气屏障、线粒体、板层小体等改变均有明显改善.荧光显微镜观察到Hoechst 33342标记的MSCs在肺内定植,且分化成大量新生血管并形成侧支循环,肺动脉重构得到有效逆转.结论 MSCs移植可有效减轻并逆转肺动脉重构的进程,其作用机制是MSCs分化形成新生血管,建立了侧支循环,从而修复野百合碱诱导的肺损伤.     

Hematologic and Bone Marrow Changes in Children with Protein-Energy Malnutrition

Background: All systems in an organism are affected by protein-energy malnutrition (PEM), but one of the worst affected is the hematopoietic system. Today PEM remains a very serious problem in developing countries. We examined the relationships between clinical features, hematological, and bone marrow changes with severe PEM from Turkey. Method: We evaluated 34 (11 females and 23 males) consecutive cases of severe PEM, with no underlying diseases aged 3–20 months. The clinical nutritional conditions of the patients were determined using the Wellcome-Trust PEM classification. Ten of the patients were in the Marasmic-Kwashiorkor (M-K) group, 10 were in the Kwashiorkor (KW) group, and 14 were in the Marasmic (M) group. Full blood count, protein, albumin, serum iron (SI), iron-binding capacity (TIBC), ferritin, vitamin B12, folic acid, complement-3 (C3), complement-4 (C4), and bone marrow were investigated in all groups. Results: Anemia was detected in 97% of patients. We determined serum iron levels were low in 67.6% of the patients, TS levels were low in 76.4% of the patients and ferritin levels were low in 20.5%. The level of vitamin B12 was normal in all patients. Bone marrow analysis showed erythroid series hypoplasia in 28.5% of patients in the M group, 50% in the KW group, and 30% in the M-K group. Marrow iron was absent in 58.8% of patients. Conclusion: The most common hematologic change in the children with PEM was anemia and major cause of anemia was iron deficiency in this study. Patients with severe PEM have normal Vit B12 and serum folate levels. Most of the patients with severe PEM had normal cellularity with megaloblastic and dysplastic changes in bone marrow due to the inadequate and imbalanced intake of protein and energy.