再生障碍性贫血患者骨髓间充质干细胞生物学特性的初步研究

目的:研究再生障碍性贫血(aplasticanemia,从)患者骨髓间充质干细胞(mesenchymalstemcells,MSCs)的生物学特性和初步探讨其异常和AA发生的可能关系。方法:取AA患者骨髓间充质干细胞,测定其生长曲线和倍增时间;流式细胞仪检测其细胞周期和免疫表型;体外定向诱导其向脂肪、成骨、内皮和神经细胞分化;用real-timePCR及油红O染色法比较AA和正常对照组MSCs的成脂分化的不同。结果:AA患者和正常成人的MSCs均呈梭形贴壁生长;AA组细胞倍增时间长于对照组;CD105、CD44、CD29、CD106、FlK-1均阳性;96．51％的细胞处在G0／G1期;AA患者的MSCs保持了多向分化潜能,体外诱导形成脂滴较对照组早,诱导早期的脂蛋白脂酶表达增高。结论:再生障碍性贫血患者的骨髓间充质干细胞增殖能力较正常成人弱,骨髓间充质干细胞的易成脂性可能参与了再障的发病环节。     

骨髓间充质干细胞在移植免疫方面的研究进展

骨髓间充质干细胞(Mesenchymal stem ells,MSCs)是存在于骨髓中一类低免疫原性的非造血成体干细胞,体外研究表明MSCs能够通过抑制混合淋巴细胞反应抑制抗原呈递细胞分化成熟及功能发挥、抑制CTL形成、抑制NK细胞活性、增加调节性T细胞比例等途径发挥免疫调节作用。体内实验证明,MSC输注能够延长狒狒异体皮肤移植的存活时间,而在小鼠心脏移植的模型中,体外诱导免疫耐受的MSCs在活体内反而加速了小鼠的排斥反应,临床上输注MSCs可缓解移植物抗宿主病(GVHD)。本文对MSCs的免疫学特性及免疫调控功能的研究进展作一综述。     

骨髓间充质干细胞对再生障碍性贫血患者FoxP3基因表达的影响研究

目的研究骨髓间充质干细胞(MSC)对再生障碍性贫血(AA)患者外周血Fox P3m RNA表达量的影响,并初步探讨MSC发挥作用的机制。方法分离AA患者外周血T淋巴细胞,与体外培养扩增的健康供者MSC按3:1比例行共培养,共培养方式分为直接接触法及Transwell法,共培养72 h后收集T淋巴细胞,提取细胞总RNA,逆转录成c DNA,实时定量PCR检测共培养前后Fox P3m RNA的相对表达量。结果 T淋巴细胞与MSC共培养72 h后Fox P3m RNA相对表达量较前升高,直接接触组为9.13,Transwell组为5.18。结论 MSC可通过与T细胞直接接触或旁分泌机制上调AA患者外周血T细胞的Fox P3m RNA表达。     

再生障碍性贫血中残存造血细胞的生物学特征

再生障碍性贫血是T细胞免疫介导的骨髓衰竭,造血前体细胞在淋巴细胞及细胞因子作用下凋亡增加.再生障碍性贫血不仅造血干/祖细胞池萎缩,其残存造血细胞还存在功能缺陷,与老化造血干细胞特征相似.在免疫攻击和造血压力作用下,残存造血细胞增殖能力减退、对细胞因子反应不良,端粒缩短、遗传不稳定,或虽逃逸免疫攻击,发生克隆性造血和向M...     

再生障碍性贫血中医药防治研究进展

再生障碍性贫血是由多种原因引起的骨髓造血功能衰竭,以全血细胞减少为特征的一种综合病症。目前普遍认为其发病机理与免疫因素介导的造血干细胞受损导致造血微环境异常相关。近年来,中医药以其独特的理论体系和治疗方法,在再生障碍性贫血的病因病机及防治防等研究方面取得了较大进展。中医药防治再生障碍性贫血一般从肾精亏虚、毒损髓络、生血乏源入手,以补肾填精、生血解毒为治疗法则,采用中药方剂或/和联合西药,可提高治疗有效率,降低西药的毒副作用,并可以调控T淋巴细胞的分化及相关细胞因子的表达。中药治疗可以改善骨髓造血微环境、不同程度的恢复骨髓的造血功能,提高患者的生存质量。现就中医药对此病的相关研究作一综述。     

白细胞介素21的生物学功能及其抗肿瘤效应

白细胞介素21（IL-21）属于I类细胞因子,主要由活化的CD4＋T产生,作用于免疫系统中的大部分骨髓、淋巴细胞,具有广泛的生物学功能,是联系主动免疫与被动免疫的中间因子。IL-21能够调节体液和细胞介导的免疫应答,促进T细胞增殖分化,调节B细胞的增殖分化与调亡,增强NK细胞的细胞毒性作用与免疫监督功能。此外,IL-21还具有抗肿瘤作用,与其他细胞因子、疫苗等联合应用,可增强其抑制肿瘤细胞生长的功能。该文综述了IL-21的生物功能及其应用于抗肿瘤治疗的研究前景。     

间充质干细胞表达趋化因子及受体的相关生物学功能

间充质干细胞(mesenchymal stem cells,MSCs)是一群存在于骨髓间质和其他组织间质的干细胞,表达CD34和CD133.近来研究发现,存在于骨髓的间充质干细胞除了能支持造血,向骨细胞、软骨细胞和脂肪细胞进行多向分化外,其分泌的趋化因子及其相关受体在MSCs的信号转导、维持内环境的稳定、损伤修复、免疫调节、支持造血等功能中也发挥了关键性的作用.     

问充质干细胞体外调控骨髓造血前体细胞向单核系分化

研究间充质干细胞(MSC)能否在体外调控造血.体外分离培养人骨髓来源的MSC,RT-PCR检测其造血生长因子的表达,并以其为饲养层细胞,接种骨髓单个核细胞(MNC),观察生长情况,并通过形态学观察和流式细胞术分析,鉴定细胞来源和分化方向.结果显示,MSC构成性表达SCF、Flt3L和M-CSF,不表达G-CSF和GM-CSF,在骨髓MNC和MSC共培养体系中,大约2周左右可以看到大量的圆形细胞粘附在梭型MSC上生长,细胞胞体为圆形,胞浆较丰富,胞核为圆形、半月型或肾型,部分细胞呈典型的单核细胞形态,流式细胞术分析该类细胞表达CD14,不表达CD15、CD41、glycophorin A、CD5和CD19.表明不需要添加外源性造血生长因子,间充质干细胞能在体外调控骨髓造血前体细胞向单核系分化,其定向分化可能与MSC分泌造血生长因子及MSC与造血细胞间相互作用有关.     

间充质干细胞体外调控骨髓造血前体细胞向单核系分化

研究间充质干细胞(MSC)能否在体外调控造血。体外分离培养人骨髓来源的MSC,RT-PCR检测其造血生长因子的表达,并以其为饲养层细胞,接种骨髓单个核细胞(MNC),观察生长情况,并通过形态学观察和流式细胞术分析,鉴定细胞来源和分化方向。结果显示,MSC构成性表达SCF、Flt3L和M-CSF,不表达C-CSF和GM-CSF,在骨髓MNC和MSC共培养体系中,大约2周左右可以看到大量的圆形细胞粘附在梭型MSC上生长,细胞胞体为圆形,胞浆较丰富,胞核为圆形、半月型或肾型,部分细胞呈典型的单核细胞形态,流式细胞术分析该类细胞表达CDl4,不表达CDl5、CD41、glycophorin A、CD5和CDl9。表明不需要添加外源性造血生长因子,间充质干细胞能在体外调控骨髓造血前体细胞向单核系分化,其定向分化可能与MSC分泌造血生长因子及MSC与造血细胞间相互作用有关。     

人DC体外分化成熟特性及抗P-选择素功能域单抗对其干预调节

结合树突状细胞(DC)生物学特性, 探讨抗P-选择素lectin-EGF功能域单抗(PsL-EGFmAb)对体外培养人DC成熟和功能干预调节的作用. 通过SCF, GM-CSF, TGF-β₁, Flt-3L及TNF-α体外培养体系, 从脐血CD34⁺造血干细胞中诱导扩增获得DC, 并于细胞成熟过程中用PsL-EGFmAb及辅以IL-10作为对照进行干预. 分别观察和检测DC形态学及细胞活力, 细胞表面分子HLA-DR, CD1a, CD11c, CD54, CD83, CD80, CD86, CD209(DC-SIGN)及CD62P, E, L(P-、E-、L-选择素)表达, 细胞内活性氧(ROS)水平, 及IL-12p35, p40 mRNA与NF-κBP50, P65 mRNA表达, 培养上清液中IL-12p70分泌含量, 以及DC体外对T淋巴细胞刺激能力, 以此分析PsL-EGFmAb 对DC成熟与功能的干预状况. 结果显示, 未成熟DC高表达属模式识别受体的C型凝集素DC-SIGN外, 且胞内蓄积适量ROS, 具备了细胞吞噬能力. 成熟DC除仍高表达DC-SIGN, 伴随细胞内NF-κB基因明显表达, 其表面黏附共刺激分子CD11c, CD83, CD80, CD86表达上调, 且细胞因子IL-12合成分泌增加, 并具明显的体外刺激T淋巴细胞增殖能力, 符合于抗原提呈细胞特征. 此外, 未成熟和成熟DC基本不表达P-, E-选择素, 而分别高表达和低表达L-选择素. 进一步发现, PsL-EGFmAb较对照IL-10对DC表面DC-SIGN表达有抑制作用; 也能抑制细胞内NF-κB基因表达, 并相应抑制或下调DC黏附共刺激分子CD11c, CD83, CD80, CD86及HLA-DR表达, 抑制IL-12基因转录及其合成分泌, 以及抑制DC体外刺激T细胞增殖的能力. 上述结果表明, PsL-EGFmAb对DC分化成熟及功能具有抑制作用, 提示此作用与其抑制作为DC模式识别受体及功能分子DC-SIGN有关, 并可能是通过影响NF-κB信号途径起作用.     

Immune recovery after allogeneic stem cell transplantation: study of 19 patients

The aim of this study was to access average delays for novogeneration of myeloid and lymphoid cells after allogeneic bone marrow transplantation (BMT) outcome and factors affecting this organization. A prospective analysis over 2 years (01/01/07 to 31/12/08) enrolling 19 children treated with allogeneic intrafamilial bone marrow transplantation. Indications for bone marrow transplantation were: aplastic anemia (3 cases), bemoglobinopathies (9 cases), myelodysplastic syndrome (1 case) and primary immunodeficiency (6 cases). Different conditioning regiments were used according to the indication. The study of immune reconstitution was based on the quantitative determination of immunoglobulin and lymphocyte subpopulation. These tests were routinely requested to 1 month, 2 months, 3 months, 6 months, 9 months and 12 months. The average time of engraftment was 18 days (12-24). A rate of CD4+T lymphocytes>200/mm3 was provided within an average of 2,5 months (1-7). The average time to obtain CD8+T lymphocytes>200/mm3 was 2 months (1-5). The humoral immune reconstitution was made within an average of 2 months (1-4). A report of CD4+/CD8+T lymphocytes>I was obtained within 10 months and a half (1-24). Univaried analysis showed a correlation between the bone marrow sex matched and the faster reorganization of CD8+T cells (p=0.042). A quantity of CD34+>6 10(6)/kg was significantly associated with the recapture of a formula lymphocyte CD4+/CD8+T>1 (p=0.03) Immune recovery post bone marrow transplantation in children begins with myeloid lineage then lymphoid B then lymphoid T The inversion of the report CD4+/CD8+T lymphocytes, seems to be influenced by the high contain of CD34+cells in the graft as well as the type of conditioning.     

Increased Bone Marrow (BM) Plasma Level of Soluble CD30 and Correlations with BM Plasma Level of Interferon (IFN)-γ, CD4/CD8 T-Cell Ratio and Disease Severity in Aplastic Anemia

Idiopathic aplastic anemia (AA) is an immune-mediated bone marrow failure syndrome. Immune abnormalities such as decreased lymphocyte counts, inverted CD4/CD8 T-cell ratio and increased IFN-γ-producing T cells have been found in AA. CD30, a surface protein belonging to the tumor necrosis factor receptor family and releasing from cell surface as a soluble form (sCD30) after activation, marks a subset of activated T cells secreting IFN-γ when exposed to allogeneic antigens. Our study found elevated BM plasma levels of sCD30 in patients with SAA, which were closely correlated with disease severity, including absolute lymphocyte count (ALC) and absolute netrophil count (ANC). We also noted that sCD30 levels were positively correlated with plasma IFN-γ levels and CD4/CD8 T-cell ratio in patients with SAA. In order to explain these phenomena, we stimulated T cells with alloantigen in vitro and found that CD30⁺ T cells were the major source of IFN-γ, and induced CD30⁺ T cells from patients with SAA produced significantly more IFN-γ than that from healthy individuals. In addition, increased proportion of CD8⁺ T cells in AA showed enhanced allogeneic response by the fact that they expressed more CD30 during allogeneic stimulation. sCD30 levels decreased in patients responded to immunosuppressive therapy. In conclusion, elevated BM plasma levels of sCD30 reflected the enhanced CD30⁺ T cell-mediated immune response in SAA. CD30 as a molecular marker that transiently expresses on IFN-γ-producing T cells, may participate in mediating bone marrow failure in AA, which also can facilitate our understanding of AA pathogenesis to identify new therapeutic targets.     

Long-lasting inhibitory effects of fetal liver mesenchymal stem cells on T-lymphocyte proliferation

Human bone marrow mesenchymal stem cells (BM-MSC) are multipotent progenitor cells that have transient immunomodulatory properties on Natural Killer (NK) cells, Dendritic Cells (DC), and T cells. This study compared the use of MSC isolated from bone marrow and fetal liver (FL-MSC) to determine which displayed the most efficient immunosuppressive effects on T cell activation. Although both types of MSC exhibit similar phenotype profile, FL-MSC displays a much more extended in vitro life-span and immunomodulatory properties. When co-cultured with CD3/CD28-stimulated T cells, both BM-MSC and FL-MSC affected T cell proliferation by inhibiting their entry into the cell cycle, by inducing the down-regulation of phospho-retinoblastoma (pRb), cyclins A and D1, as well as up-regulating p27(kip1) expression. The T cell inhibition by MSC was not due to the soluble HLA-G5 isoform, but to the surface expression of HLA-G1, as shown by the need of cell-cell contact and by the use of neutralizing anti-HLA-G antibodies. To note, in a HLA-G-mediated fashion, MSC facilitated the expansion of a CD4(low)/CD8(low) T subset that had decreased secretion of IFN-γ, and an induced secretion of the immunomodulatory cytokine IL-10. Because of their longer lasting in vitro immunosuppressive properties, mainly mediated by HLA-G, and their more efficient induction of IL-10 production and T cell apoptosis, fetal liver MSC could be considered a new tool for MSC therapy to prevent allograft rejection.     

Yin and Yang of mesenchymal stem cells and aplastic anemia

Acquired aplastic anemia(AA) is a bone marrow failure syndrome characterized by peripheral cytopenias and bone marrow hypoplasia. It is ultimately fatal without treatment, most commonly from infection or hemorrhage. Current treatments focus on suppressing immune-mediated destruction of bone marrow stem cells or replacing hematopoietic stem cells(HSCs) by transplantation. Our incomplete understanding of the pathogenesis of AA has limited development of targeted treatment options. Mesenchymal stem cells(MSCs) play a vital role in HSC proliferation; they also modulate immune responses and maintain an environment supportive of hematopoiesis. Some of the observed clinical manifestations of AA can be explained by mesenchymal dysfunction. MSC infusions have been shown to be safe and may offer new approaches for the treatment of this disorder. Indeed, infusions of MSCs may help suppress auto-reactive, T-cell mediated HSC destruction and help restore an environment that supports hematopoiesis. Small pilot studies using MSCs as monotherapy or as adjuncts to HSC transplantation have been attempted as treatments for AA. Here we review the current understanding of the pathogenesis of AA and the function of MSCs, and suggest that MSCs should be a target for further research and clinical trials in this disorder.     

Anti-thoracic duct lymphocyte globulin stimulates human megakaryocytopoiesis in vitro

Anti-thoracic duct lymphocyte globulin (ALG) therapy is effective in patients with aplastic anemia. We examined the effect of ALG on human megakaryocyte progenitor cells (colony-forming unit-megakaryocyte, CFU-Meg) in vitro. Normal human bone marrow mononuclear cells (MNC) were cultured in plasma clots with varying concentrations of ALG or non-immunized horse IgG. After 12 days of culture, significant megakaryocyte colony formation was observed in cultures containing ALG but not in cultures containing non-immunized horse IgG. The peak stimulatory effect seemed to occur with 10-25 micrograms/ml of ALG. When marrow MNC, depleted of adherent and T cells, were cultured in plasma clots with ALG, its stimulatory effect on megakaryocytopoiesis decreased markedly. Finally, it was demonstrated that ALG stimulated marrow MNC to produce a factor stimulatory for CFU-Meg. The in vitro megakaryocytopoietic stimulatory effect of ALG may be related to its clinical efficacy in some patients with aplastic anemia.     

The Clinical and Immune Characteristics of Patients with Hepatitis-Associated Aplastic Anemia in China

Hepatitis-associated aplastic anemia (HAAA) is a variant of severe aplastic anemia (SAA) in which bone marrow failure follows an acute attack of hepatitis. Its pathogenesis is poorly understood. We investigated the prevalence of HAAA among cases of newly diagnosed SAA presenting to our hospital between January 1998 and February 2013, and analyzed the clinical and immune characteristics of HAAA and non-hepatitis-associated SAA (non-HASAA) patients. The prevalence of HAAA among cases of SAA was 3.8% (36/949), and the majority of patients (33/36) were seronegative for a known hepatitis virus. Compared with non-HASAA patients, HAAA patients had a larger proportion of CD8+ T cells, a lower ratio of CD4+/CD8+ T cells, and a smaller proportion of CD4+CD25+ regulatory T cells. There was no significant difference in peripheral blood count, bone marrow cellularity, or the number of blood transfusions received between HAAA and non-HASAA patients. HAAA patients had a higher early infection rate and more infection-related mortality in the first 2 years after diagnosis than non-HASAA patients, and their 2-year survival rate was lower. The results demonstrate that HAAA patients have a more severe T cell imbalance and a poorer prognosis than non-HASAA patients.     

Lymphocytes and lymphokines in aplastic anemia: pathogenic role and implications for pathogenesis

Clinical observations first suggested an immune basis for aplastic anemia. Laboratory studies have implicated a suppressor T lymphocyte producing molecules like gamma-interferon that inhibit hematopoiesis. Extrapolation from in vitro studies is complicated by synergy and antagonism among lymphokines and interleukins, the presence of regulators of proliferation produced by bone marrow cells, and possible differences between endogenous and exogenously administered molecules on cell growth.     

Mesenchymal stromal cells augment CD4+ and CD8+ T-cell proliferation through a CCL2 pathway

Background aimsMesenchymal stromal cells (MSC) derived from bone marrow are immunosuppressive in vitro and in vivo. Recent evidence, however, has shown that in certain settings, MSC can also be immunostimulatory. The mechanisms involved in this process are largely unknown.MethodsMouse spleen T cells were stimulated with allogeneic mixed lymphocyte reaction (MLR) or anti-CD3/CD28 beads and treated with autologous bone marrow MSC or MSC-conditioned medium. CD4+ and CD8+ T-cell proliferation was analyzed after treatment.ResultsWe show that MSC have both suppressive and stimulatory functions toward T cells after stimulation with anti-CD3/CD28 beads or in an MLR. This depended on the ratio of MSC to responder T cells, with low numbers of MSC increasing and higher numbers inhibiting T-cell proliferation. Immunostimulatory function was mediated, in part, by soluble factors. MSC immunosuppression of the MLR was indirect and related to inhibition of antigen-presenting cell maturation. Direct effects of MSC-conditioned medium during anti-CD3/CD28 stimulated proliferation were entirely stimulatory and required the presence of the T-cell receptor. MSC supernatant contained both CCL2 and CCL5 at high levels, but only CCL2 level correlated with the ability to augment proliferation. An anti-CCL2 antibody blocked this proliferative activity.ConclusionsCCL2 plays an important role in the immunostimulatory function of MSC, and we further hypothesize that the immunomodulatory role of MSC is determined by a balance between inhibitory and stimulatory factors, suggesting the need for caution when these cells are investigated in clinical protocols.