儿童再生障碍性贫血的规范诊治现状及研究进展

再生障碍性贫血(再障)是儿童期严重的血液病之一,诊断和治疗较为困难,尤其是重型再障预后较差.我国属于再障高发地区,必须引起足够的重视.目前已经明确,T淋巴细胞"免疫介导"是再障的主要发病机制,由于T淋巴细胞功能异常,而导致骨髓造血干细胞受到抑制.成功的异基因造血干细胞移植虽能根治再障,但受到供体来源困难、医疗费用昂贵、治疗难度大和医疗风险高等限制,国内难以广泛开展.近年来,根据再障免疫介导致病机制所开展的免疫抑制治疗已经获得较为满意的疗效,是无合适供体进行造血干细胞移植的重型和难治型再障的最佳替代疗法.文章对有关再障发病机制的研究进展、诊断与分型标准、鉴别诊断要点、治疗原则与治疗方法以及疗效评价标准等进行简要介绍.     

儿童再生障碍性贫血的诊断与治疗

我国属于再生障碍性贫血(再障)高发地区,儿童期为再障好发年龄段.在目前我国儿童白血病诊治研究取得显著进展的今天,必须高度重视儿童再障的诊治研究.现归纳小儿再障的发病机制、诊断分型标准、治疗方法 和疗效标准的国内外研究进展,尤其是针对诊断分型、鉴别诊断等方面提出了需要临床重视的问题,并详细介绍了治疗方法 、药物选择原则与常见药物不良反应及其具体防治措施.     

造血因子对再生障碍性贫血的治疗作用

８０年代以来，对再生障碍性贫血（简称再障）的病因及治疗的研究进展迅速。通过建立小鼠模型的实验研究，再障的病因已拓展到基因水平，而对细胞因子，尤其是造血干细胞固子（ＳＣＦ）性质的认识及其分离、提纯和重组克隆的成功及其体内外研究，提示针对基因水平的病因治疗成为可能，各种因子对再障的治疗有很广阔的临床应用前景。     

儿童Ph-like急性淋巴细胞白血病的研究进展

费城染色体样急性淋巴细胞白血病(Ph-like ALL)是一组基因表达谱与费城染色体阳性ALL(Ph~+ALL)相似的B-ALL(B-lineage ALL)亚群,涉及一系列细胞因子受体基因及激酶信号通路异常活化的相关基因改变,并常伴淋系发育相关转录因子异常。Ph-like ALL在高危组儿童B-ALL的比例高达15%,其临床特征与不良预后相一致。酪氨酸激酶抑制剂(TKIs)联合化疗显著改善儿童Ph~+ALL预后提示基于Ph-like ALL分子遗传学异常的精准靶向治疗具有良好的研究前景。该文结合近年Ph-like ALL的相关研究进展,对儿童Ph-like ALL的基因改变及发病机制、临床特征、诊断及治疗进行综述。     

造血因子对再生障碍性贫血的治疗作用

８０年代以来，对再生障碍性贫血（简称再障）的病因及治疗的研究进展迅速。通过建立小鼠模型的实验研究，再障的病因已拓展到基因水平，而对细胞因子，尤其是造血干细胞因子（ＳＣＦ）的性质的认识及其分离、提纯和重组克隆的成功及其体内外研究，提示针对基因水平的病因治疗成为可能，和种因子对再障的治疗有很广阔的临床应用前景。     

儿童急性非淋巴细胞白血病染色体与临床预后分析

目的 研究儿童急性非淋巴细胞白血病(ANLL)染色体变化及预后。方法 通过短期培养法对43例ANLL患儿骨随细胞进行染色体核型分析，并观察临床疗效。结果 43例ANLL患儿染色体核型异常检出率为74％(32／43)，结构异常与白血病类型有特殊的对应关系；其中结构异常50％，数目异常31％，数目并结构异常19％，核型异常率与性别无关(P＞0．05)；核型正常组及超二倍体组患儿缓解率高于核型异常组(P＜0．01)，核型正常、超二倍体t(15；17)易位者疗效较好，亚二倍体及t(8；21)易位者疗效较差。结论 染色体分析对指导ANLL的治疗及预后判断具有重要作用。     

4例Williams-Beuren综合征家系遗传学诊断与产前诊断

Williams-Beuren综合征是一类常见的染色体微缺失综合征,早期诊断及干预对患儿及其家庭十分重要。本研究应用染色体核型分析技术(G显带),多重连接依赖探针扩增技术(MLPA)及微阵列比较基因组杂交技术(array-CGH)对4个家系中1例超声异常的胎儿及3例发育异常患儿行染色体核型和基因组DNA分析,为这4个家庭的再生育提供指导,为产前诊断提供依据。研究结果发现1例产前超声异常孕妇羊水及3例发育异常患儿外周血MLPA分析提示染色体7q11.23区域ELN基因探针信号降低,array-CGH检测提示染色体7q11.23区域杂合缺失。4个家系中母亲再次妊娠时取羊水细胞标本行上述检测均未发现异常。研究结果提示MLPA技术及array-CGH技术能够快速、准确地诊断Williams-Beuren综合征,为临床提供更好的遗传咨询服务。     

儿童慢性嗜酸粒细胞白血病1例并文献复习

目的通过分析一例嗜酸细胞异常增高的儿童慢性嗜酸粒细胞白血病,结合文献阐明该病的临床特点、细胞遗传学改变、诊断、治疗及预后。方法临床病例分析及文献综述。结果该病人及文献复习显示本病临床特点主要为贫血、出血、肝脾淋巴结肿大以及嗜酸细胞破坏后释放出大量细胞因子引起全身各脏器损害的相关症状;细胞遗传学改变通常为涉及血小板衍生的生长因子受体基因的染色体异位及Wilms瘤基因的高表达;与特发性嗜酸细胞增生症的鉴别主要为细胞遗传学的改变,前者多有克隆性染色体异常以及癌基因的的异常表达;治疗除化疗外还有IFN-α的免疫治疗,酪氨酸激酶抑制剂靶向治疗以及干细胞移植;该病预后不佳。结论:该病在儿童非常罕见,除白血病常见症状外应重视大量细胞因子引起的各脏器相关症状,确诊主要靠细胞遗传学,治疗方法虽多但疗效多不确切,干细胞移植是目前较为理想的方法,在治疗过程中辅以白细胞分离和血浆置换对于防止嗜酸细胞破坏后大量细胞因子对各脏器的损害十分必要。     

T细胞在再生障碍性贫血发病机制中的作用

再生障碍性贫血(再障)是多种病因导致的以骨髓衰竭伴全血细胞减少为特征的疾病,其发病机制与免疫有着密切的关系.细胞免疫介导的造血损伤可能是再障的主要发病机制之一,而T细胞作为细胞免疫中的主要效应细胞,在骨髓及外周血中均有异常,如CD4+和CD8+ T细胞数量与亚群的变化、T细胞受体表达状态异常等均与该病的发生有关.再障患者淋巴细胞凋亡状态和白细胞介素-2、γ干扰素等细胞因子的差异也可能是该病发生和发展的重要原因.近来研究发现T细胞与骨髓问充质干细胞之间的作用也在其免疫发病机制中起着重要的作用.     

Wiskott-Aldrich综合征高危儿七例产前诊断

目的 探讨Wiskott-Aldrich综合征(WAS)高危孕妇羊水脱落细胞基因分析及脐静脉穿刺脐血WAS蛋白(WASP)检测在WAS高危儿产前诊断中的意义.方法 2008年至2010年我院经WASP流式检测和基因分析明确诊断的7例WAS患儿.详细记录先证者病史,进行家族相关亲属基因检测,建立7个WAS家系图谱.2008年至2011年对7个家系中携带异常基因的7个高危孕妇于孕18 ～ 20周经羊膜腔穿刺抽取羊水.其中部分羊水提取细胞DNA,经PCR扩增WAS基因.PCR产物进行双向序列重复测定.其中1例高危孕妇孕28周采集脐带血,采用流式细胞术检测WASP.培养羊水中胎儿脱落细胞,采用原位制片及G带染色技术进行染色体核型分析.产后采集高危儿外周血进行基因分析和WASP检测验证产前诊断结果.结果 7例WAS高危儿羊膜腔穿刺均成功,羊水细胞培养成功率100％.WAS基因和染色体核型分析结果显示1例正常男性胎儿,4例正常女性胎儿,2例为女性异常基因携带者.1例脐带血流式细胞术检测WASP显示正常表达.7例高危儿均顺利出生,产后WASP和基因分析结果均正常,与产前结果相同.结论 羊水脱落细胞核型、基因分析与脐带血WASP检测可为WAS高危孕妇提供可靠的产前诊断服务.     

小儿再生障碍性贫血疗效总结

目的总结72例小儿再生障碍性贫血(以下简称再障)的疗效。方法72例患儿中30例慢性再障,42例急性再障。慢性再障给予安雄、CsA治疗,急性再障给予安雄、CsA、大剂量丙种球蛋白、ATG治疗,其中2例接受异基因造血干细胞移植。结果72例再障患儿中慢性再障组和急性再障组在性别和发病年龄上无明显差异(P>0.05)。30例慢性再障病人中17例进步或治愈,11例无效或进展,无死亡病例;42例急性再障病人中14例进步或治愈,20例无效或进展,7例死亡,两组病人达进步或治愈者均无复发病例,两组总有效率有显著差异(56.67%vs33.33%P<0.05),两组病人总的生存率统计学上有差异(100%vs83.33%P<0.05)。两组出现疗效的时间无差异(P>0.05),而在中位输血时间间隔上,两组有显著差异(P<0.005)。在42例急性再障病人中,28例接受ATG治疗,14例未接受ATG治疗,ATG组中有11例出现进步或治愈,未接受ATG组中仅3例出现疗效,两组有明显统计学差异(42.13%vs21.43%P<0.05),72例再障病人中有3例转变为AML(分别为M2,M3,M5),2例接受了异基因造血干细胞移植后治愈。结论慢性再障治疗疗效及生存率明显高于急性再障,而联合免疫治疗是治疗急性再障除了骨髓移植外的最有效的方法。     

Dyskeratosis congenita and telomerase

PURPOSE OF REVIEW: Dyskeratosis congenita, a rare condition characterized by mucocutaneous abnormalities and bone marrow failure, is caused by inherited defects in the telomerase complex. Autosomal dominant dyskeratosis congenita is associated with mutations in the RNA component of telomerase, hTERC, while X-linked dyskeratosis congenita is due to mutations in the gene encoding dyskerin, a protein implicated in both telomerase function and ribosomal RNA processing. This review highlights recent research on dyskeratosis congenita and its relevance to other fields, including cancer and aging. RECENT FINDINGS: Newly developed animal models suggest that defects in ribosomal RNA processing contribute to the phenotype of X-linked dyskeratosis congenita. Bone marrow dysfunction may be the first manifestation of dyskeratosis congenita in children, and hTERC mutations have been detected in a subset of patients presumed to have idiopathic aplastic anemia or myelodysplastic syndrome. In vitro studies suggest that hTERC mutations associated with dyskeratosis congenita or aplastic anemia either impair the specific activity of telomerase, decrease hTERC stability, or disrupt assembly of the telomerase complex. Recent clinical reports suggest that nonmyeloablative conditioning regimens afford better outcomes in patients with dyskeratosis congenita who require hematopoietic stem cell transplantation. SUMMARY: Studies of dyskeratosis congenita have shed light on the pathobiology of aplastic anemia and other forms of bone marrow dysfunction. It seems likely that mutations in other genes involved in telomere maintenance will be linked to bone marrow failure or other human diseases. Genetic testing for occult dyskeratosis congenita may be warranted in selected patients with aplastic anemia or myelodysplastic syndrome, as this may impact the choice of therapies.     

Treatment of aplastic anemia in children by bone marrow transplantation (author's transl)]

The course of a successful bone marrow transplantation in a 8 year old boy with severe aplastic anemia is presented. The boy lives now 22 months after bone marrow transplantation with normal bone marrow function and an essentially undisturbed performance; The indication for bone marrow transplantation for severe aplastic anemia in children is discussed. Despite improved intensive supportive care (antibiotics, infectious prophylaxis, substitution of blood components) and the treatment with corticosteroids and/or androgens the mortality of sever aplastic anemia in childhood is still high. The advantages of early bone marrow transplantation in severe cases are stressed.     

儿童再生障碍性贫血的分型研究

目的探讨儿童再生障碍性贫血(再障)的分型标准。方法对已经确诊的61例儿童急性再障和45例儿童慢性再障病例进行回顾性分析。结果急性再障患儿有如下特点外周血呈全血细胞减少,淋巴细胞比例明显增高;髂骨及胸骨骨髓呈多部位增生不良,造血细胞明显减少,非造血细胞明显增多,巨核细胞明显减少,小粒细胞面积＜50％,细胞成分以非造血细胞为主;增生活跃患儿的骨髓中,非造血细胞比例明显增多,巨核细胞明显减少。慢性再障患儿的特点是外周血呈血小板减少和(或)白细胞减少和(或)贫血的血象,淋巴细胞比例增高;髂骨骨髓以增生活跃为主,但粒细胞系比例减低,红细胞系比例可正常,非造血细胞比例增高,巨核细胞减少,小粒细胞面积＜50％,细胞成分以非造血细胞为主。结论儿童急性再障的表现与Camitta提出的重型再障的诊断标准相似,而儿童慢性再障的表现与Camitta提出的轻型再障不同,根据本文结果提出儿童再障的分型标准及胸骨骨髓在诊断急、慢性再障中的意义。     

Low Expression of Basic Fibroblastic Growth Factor in Mesenchymal Stem Cells and Bone Marrow of Children with Aplastic Anemia

Background: Our previous experiments with gene chip suggested that basic fibroblastic growth factor (FGF2) levels were lower in mesenchymal stem cell (MSC) from aplastic anemia patients. The purpose of this study was to determine the expression of FGF2 in MSC and in bone marrow of children with aplastic anemia to better understand the role of low FGF2 expression in the pathogenesis of aplastic anemia. Procedure: MSCs from the bone marrow of aplastic anemia children and control group were cultured in vitro. Growth curves of primary and passage MSC were plotted. FGF2 gene expression in MSCs was detected using quantitative real-time polymerase chain reaction (RT-PCR). FGF2 protein expression in mononuclear cells and FGF2 protein level in extracellular fluid of bone marrow were also investigated. Result: Decreased growth of MSCs from aplastic anemia children was observed after passage 8 in serial subcultivation, and FGF2 gene expression was downregulated. Within the patients’ bone marrow, low FGF2 expression was validated both in mononuclear cells and in the extracellular fluid. Conclusion: Low FGF2 gene expression in MSCs and low FGF2 protein level in bone marrow of aplastic anemia may involve to pathogenesis of aplastic anemia.     

Screening for paroxysmal nocturnal hemoglobinuria (PNH) clone in Egyptian children with aplastic anemia

Aplastic anemia and paroxysmal nocturnal hemoglobinuria (PNH) are clinically related. In addition to their concurrent or sequential appearance in individual patients, PNH and aplastic anemia share several pathophysiologic features. The aim of the present study was to screen for PNH clone in Egyptian aplastic anemia pediatric patients before the initiation of any specific therapy and to evaluate the clinical status of studied patients 3-6 months after initiation of immunosuppressive therapy. We studied 11 pediatric patients with newly diagnosed acquired aplastic anemia and followed them up clinically for 3-6 months after initiation of immunosuppressive therapy. In addition to routine clinical and laboratory evaluation, sucrose lysis test and staining of bone marrow for CD59 were performed in all subjects. All studies cases had severe aplastic anemia (SAA) except one case which had very severe aplastic anemia (VSAA). Sucrose lysis test was negative in all studied cases. Presence of PNH clone (as evident by loss of normal staining of hematopoietic cells for CD59 = CD59 negative cells) was evident in four subjects. All cases with PNH clone were >6 years old and one of them developed splenic vein thrombosis. As regards the laboratory data WBC < or = 2.8 x 10(3)/mm3 and reticulocytes > or = 0.6 per cent were the most frequent factors associated with PNH clone found in all PNH subjects, but only in 28.6 per cent and 14.3 per cent respectively, of non-PNH subjects. Mortality rate was higher in non-PNH subjects (28.5 per cent) compared to 25 per cent of PNH subjects. We conclude that immunohistochemical staining of bone marrow sections is a sensitive tool to detect the emergence of PNH clone in aplastic anemia patients. Thrombotic complications should be anticipated in cases with aplastic anemia having a PNH clone.     

Immunotherapy for severe aplastic anemia following orthotopic liver transplantation in children

Severe aplastic anemia is a well-recognized complication of fulminant non-A, non-B, and non-C hepatitis requiring orthotopic liver transplantation. The first line of therapy for cure in the treatment of aplastic anemia is a histocompatible bone marrow transplant. Immunosuppressive therapy is also effective if a histocompatible donor is not available. We describe two children who developed severe aplastic anemia following orthotopic liver transplant who achieved bone marrow recovery with a single course of anti-thymocyte globulin, solumedrol, and adjustments to their immunosuppressive therapy for prevention of liver allograft rejection.     

Etiologic mechanisms of hematopoietic failure

Our understanding of the pathophysiology of aplastic anemia has lagged behind advances in treatment of the disease. The data available indicate that the heterogeneous assortment of drugs, as well as the array of chemical, physical, and infectious agents that are clinically associated with aplastic anemia, probably exert their action through restricted pathways. Based on certain clinical features of aplastic anemia, animal models of hematopoietic failure, and considerations of the organization of the hematopoietic system, we propose two general types of aplastic anemia as a conceptual framework for further studies: type I aplastic anemia, representing a congenital or acquired stem cell defect that can only be cured by bone marrow transplantation, and type II aplastic anemia, which is caused by reversible suppression of normal stem cells by external agents and is amenable to immunosuppressive therapy.     

抗胸腺球蛋白/抗淋巴细胞球蛋白治疗重型再生障碍性贫血的疗效及其并发症

目的：目前重型再生障碍性贫血的主要治疗手段之一是免疫抑制治疗,而抗胸腺球蛋白/抗淋巴细胞球蛋白(ATG/ALG)是其中重要的药物。该文通过回顾性分析临床资料,探讨ATG/ALG治疗重型再生障碍性贫血的疗效及其并发症的防治。方法：对1994年12月至2005年9月收治的28例诊断为重型再生障碍性贫血并接受ATG/ALG治疗的患儿的临床资料进行分析。结果：28例患儿中,基本治愈2例(7.1%),缓解4例(14.3%),进步12例(42.9%),总有效率64.3%。19例出现血清病样反应,主要表现发热9例,皮疹12例,关节痛7例,肌肉痛7例,关节肿胀3例。发生时间为用药后5~17d,持续时间1~15d,平均4.4d。3例轻症者未经处理自行缓解。其余16例给予甲基泼尼松龙每日10mg/kg,静脉推注每天1次,连用3~5d,症状均消失。3例于停用甲基泼尼松龙2~4d后再次出现血清病症状,再次给予甲基泼尼松龙后症状消失。无血清病及血清病反应轻微者ATG/ALG疗效明显优于血清病反应重者(P<0.05)。结论：ATG/ALG治疗重型再生障碍性贫血疗效肯定,血清病为治疗中常见的不良反应,应用甲基泼尼松龙3~5d可较好控制血清病症状。     

Glutathione S transferase theta 1 gene (GSTT1) null genotype is associated with an increased risk for acquired aplastic anemia in children

Two main factors have been implicated in the mechanism underlying the pathogenesis of acquired aplastic anemia: environmental factors and genetic susceptibility. Individuals vary in their ability to metabolize several DNA-damaging agents due to polymorphisms of biotransforming enzymes. Genetically determined differences in the expression of these enzymes could explain interindividual risks in developing acquired aplastic anemia. The aim of the study was to characterize the genetic polymorphism of biotransforming phase I (p450-cyp2E1) and phase II [microsomal epoxide hydrolase (mEh), glutathione S-transferase (GST)] enzymes in pediatric patients with acquired aplastic anemia. The GSTT1 null genotype (absence of both alleles) was associated with a significantly increased risk for acquired aplastic anemia (odds ratio, 2.8; 95% confidence interval, 0.15-5.7). In contrast, the GSTM1 null genotype or polymorphisms within the p450-cyp2E1 and mEh genes was not significantly different in patients and controls. Multivariate analysis was performed to assess whether the enzymes together or with other variables as age, gender, or response to therapy may have any significant association with the tested genotypes. In no combinations of the mentioned parameters was an association found with acquired aplastic anemia. GST are mainly involved in metabolizing hematotoxic and mutagenic substrates such as benzene derivatives. The GSTT1 null genotype may modulate the metabolism of exogenous pollutants or toxic intermediates. The absence of the GSTT1 enzyme, leading to genetic susceptibility toward certain pollutants, might determine the individual risk for development of acquired aplastic anemia in children.