造血干细胞表面标志物AC133的研究进展

AC133是近几年才发现的分子质量为119 ku的新型糖蛋白,主要选择性表达于成人骨髓、胎肝、脐带血及外周血等造血组织的CD34~+造血干、祖细胞(HSPC),而不表达于血管内皮细胞。AC133抗原是继CD34之后发现的又一重要的HSPC表面标志,并且可能更优于CD34原始细胞标志,具有很好的应用前景。本文就AC133的研究进展作一综述,重点介绍其抗原结构、抗体的制备和分析、表达、与其他抗原关系及应用前景。     

CD34 +自体干细胞移植联合DC-CIK细胞免疫治疗复发难治性淋巴瘤疗效观察

目的 研究CD34+自体造血干细胞移植联合树突细胞-细胞因子诱导杀伤细胞(DC-CIK)免疫治疗复发难治性淋巴瘤的临床疗效及安全性.方法 选择2011年1月-2014年5月48例复发难治性淋巴瘤患者作为研究对象,按临床治疗方法分为对照组和观察组,每组24例,对照组在淋巴瘤常规治疗基础上加用CD34+自体干细胞移植治疗,观察组在对照组基础上联合DC-CIK细胞免疫治疗.治疗结束后比较评价两组临床疗效、不良反应以及预后情况.结果 观察组临床总有效率明显高于对照组(P＜0.05).两组治疗期间不良反应发生率比较差异无统计学意义(P＞0.05).观察组平均生存时间20个月,中位生存时间18个月;对照组平均生存时间16个月,中位生存时间14个月.观察组总体生存率明显高于对照组(P＜0.05).结论 CD34+自体造血干细胞移植联合DC-CIK细胞免疫治疗复发难治性淋巴瘤患者可取得较好的临床疗效,安全性高,不良反应少,且预后效果尚可.     

Interleukin-11 enhancement of VLA-5 mediated adhesion of CD34+ cells from cord blood to fibronectin is associated with the PI-3 kinase pathway

Adhesion is required for cell growth, differentiation, survival, and function. Cell adhesion is mediated by a structurally diverse group of plasma membrane receptors, each exhibiting specialized ligand-binding properties that are needed for specific tasks. Integrin-mediated adhesion is important for hematopoietic stem (HSC)/progenitor (HPC) cell survival and may prevent programmed cell death. Interleukin (IL)-11, a multi-functional cytokine secreted by the bone marrow environment, plays an important role in regulating growth and differentiation of HSCs/HPCs. In this report, we demonstrate that IL-11 enhanced adhesion of freshly isolated and 3 day-expanded CD34+ cells to immobilized fibronectin. the expression of very late antigen (VLA)-4 and VLA-5 integrins was detected on CD34+ cells. CD34+ cells also expressed a-chain and gp130 subunits of the IL-11 receptor (R). Enhanced adhesion by IL-11 was mediated via activation of VLA-5 integrins, since this action could be blocked by monoclonal antibodies against beta 1 and alpha 5, but not alpha 4, integrins. Addition of phosphatidylinositol (PI)-3 kinase inhibitors blocked IL-11 enhanced adhesion of CD34+ cells to fibronectin. The results suggest that this enhanced adhesion is associated with the PI-3 kinase pathway, an inside-out signaling pathway.     

转HOXB4基因人骨髓MSC促进脐血CD34~+细胞体外扩增

目的 研究转HOXB4基因的人骨髓间充质干细胞(MSC)对脐血CD34~+细胞体外扩增的支持作用.方法 用病毒载体质粒转染包装细胞293T,收获病毒上清(VCM)感染MSC后,用潮霉素筛选获得转HOXB4的MSC(MSC-HOX).分别将MSC(对照组)与MSC-HOX细胞(实验组)作为CD34~+细胞体外扩增的饲养层细胞,结合细胞因子Fh3/Flk3配体(FL)、促血小板生成素(TPO)、干细胞因子(SCF)和粒细胞-集落生长因子(G-CSF)体外扩增脐血CD34~+细胞10 d,收集所有脐血细胞,检测细胞总数、CD34~+细胞总数,集落细胞形成单位(CFU)数.结果 生产重组逆转录病毒可有效将HOXB4基因转入靶细胞内并表达.脐血CD34~+细胞经体外扩增10 d后,细胞总数和CFU数两组间差异无统计学意义(P>0.05),脐血CD34~+细胞总数和比例高于对照组(P<0.05).结论 转HOXB4基因的人骨髓MSC在脐血CD34~+细胞体外扩增中,可保持CD34~+细胞未分化状态,有潜在的应用价值.     

人脐血CD34^ 内皮祖细胞的体外分化

目的：研究人脐血CD34^ 细胞群体中内皮祖细胞在体外分化为内皮细胞的过程中,干细胞标志以及内皮细胞表型随时间的变化。方法：将免疫磁珠细胞分选法（MACS）得到的CD34^ 细胞体外培养于纤连蛋白和无纤连蛋白处理的培养皿中,以免疫细胞化学鉴定贴壁细胞的内皮标志Flk-1和vWF,并以流式细胞仪分析其干细胞标志AC133。结果：贴壁细胞的内皮标志Flk-1和vWF是逐步出现的,d3时有27.0%贴壁细胞表达Flk-1,vWF不表达,d7时已100%表达vWF和Flk-1,纤连蛋白促进贴壁细胞内皮标志Flk-1和vWF的表达,d3时的表达百分率分别为34.0%和47.0%,d7时Flk-1和vWF的表达均为100%,在培养过程中,AC133阳性细胞的比例迅速下降,但纤连蛋白对AC133的表达无显著影响。结论：在内皮祖细胞分化的过程中,干细胞标志迅速消失,向内皮细胞分化,内皮细胞的表型是逐步出现的,纤连蛋白促进内皮祖细胞的分化。     

CD_7~+的急性髓细胞性白血病的临床意义

目的 :观察和探讨 CD7+ 的急性髓细胞性白血病 (AML )表达特征及它们对 AML患者预后的影响。方法 :采用间接免疫荧光法和流式细胞仪对 2 75例初诊的 AML患者进行了免疫表型检测 ,并对淋系分化抗原阳性表达率加髓系分化抗原阳性表达率大于 10 0 %患者的白血病细胞用流式细胞仪做双标记分析。结果 :2 75例 AML患者中 ,3 6例表达淋系分化抗原 CD7(占 13 .1% ) ;诱导化疗疗效方面 ,在同等化疗强度的基础上 ,CD34+ CD7+ AML患者的缓解率明显低于 CD34- CD7- AML 患者 (P<0 .0 5 )。结论 :CD7+ AML 的表达具有复杂性、异质性 ,属于一种预后不良的临床亚型 ,应进一步探索针对性的治疗策略     

rhBMP-2m对辐射小鼠骨髓CD34~+细胞变化的影响

目的　探讨小鼠受到辐射后 ,重组人骨形成蛋白成熟肽 2 (rhBMP 2m)对骨髓CD34+ 细胞变化的影响。方法　通过60 Coγ射线照射 ,建立小鼠骨髓造血损伤模型 ,经rhBMP 2m连续治疗后 ,骨髓单个核细胞计数 ,通过流式细胞仪检测骨髓单个核细胞中CD34+ 细胞比例 ,比较对照组和治疗组之间的差别。结果　经rhBMP 2m治疗的动物 ,单个核细胞数和CD34+ 细胞比例均明显高于照射对照组 ,P <0 0 5 ,n=6。结论　对于辐射引起的小鼠骨髓造血损伤 ,rhBMP 2m能够增加造血细胞数量 ,提高骨髓单个核细胞中CD34+ 细胞的比例 ,加快骨髓造血系统的重建。     

Different roles of IL-15 from IL-2 in differentiation and activation of human CD3+CD56+ NKT-like cells from cord blood in long term culture

The abundantly available source of stem cells and the low incidence of graft-versus-host disease (GVHD) made cord blood an attractive alternative source of hematopoietic stem cells for transplantation. Besides T cell and NK cell, NKT cell played an important role in low incidence of GVHD during allogeneic transplantation. IL-2 and IL-15 can stimulate T cell and NK cell proliferation, survival and activation in vitro. But they exhibited different effects on the GVHD during allogeneic transplantation. In this study, we explored the different effects of exogenous IL-2 and IL-15 on the expansion of CD3+CD56+ NKT-like cells by in vitro long term culture of cord blood mononuclear cells (CBMCs). The results showed that CD3+CD56+ NKT-like cells were derived from CD34-CD56- CBMCs and IL-2 improved CD3+CD56+ NKT-like cell expansion more strongly than IL-15. Interestingly, CD3+CD56+ NKT-like cells from IL-15-cocultured CBMCs had significantly lower apoptotic frequency and higher levels of activation markers (CD161, CD25, and IFN-gamma) than those from IL-2-cocultured CBMCs. The anti-apoptotic and activating effects of IL-15 on CD3+CD56+ NKT-like cells from CBMCs might possibly explain the pathogenic role of IL-15 in GVHD during allogeneic transplantation.     

TR短肽修饰的DSPE-PEG胶束对CD133高表达胶质瘤肿瘤干细胞体外靶向性评价(英文)

肿瘤干细胞已经成为药物递送的一个新靶点。肿瘤干细胞表面表达特异性标志物,其中CD133在胶质瘤肿瘤干细胞中高表达。因此,我们通过将特异性结合CD133的TR短肽修饰到药物递送系统上,来达到靶向递送药物的胶质瘤干细胞的目的。首先,我们将TR短肽与DSPE-PEG连接,构建包载香豆素-6的主被动DSPE-PEG胶束。之后,我们通过荧光激发免疫细胞分选法和肿瘤球培养法分离胶质瘤肿瘤干细胞。通过共聚焦显微镜和流式细胞仪检测发现,肿瘤干细胞对T R修饰的胶束摄取增多,证明TR修饰胶束具有体外靶向性。由此表明,TR修饰胶束有可能提供了针对CD133+肿瘤干细胞的新的治疗策略。     

The Hsp32 inhibitors SMA-ZnPP and PEG-ZnPP exert major growth-inhibitory effects on D34+/CD38+ and CD34+/CD38- AML progenitor cells

Heat shock protein 32 (Hsp32), also known as heme oxygenase 1 (HO-1), has recently been identified as a potential target in various hematologic malignancies. We provide evidence that Hsp32 is constitutively expressed in primary leukemic cells in patients with acute myeloid leukemia (AML) and in various AML cell lines (HL60, U937, KG1). Expression of Hsp32 mRNA was demonstrable by qPCR, and expression of the Hsp32 protein by immunocytochemistry and Western blotting. The stem cell-enriched CD34+/CD38+ and CD34+/CD38- fractions of AML cells were found to express Hsp32 mRNA in excess over normal CD34+ progenitor cells. Two Hsp32-targeting drugs, pegylated zinc-protoporphyrin (PEG-ZnPP) and styrene-maleic-acid-copolymer-micelle-encapsulated ZnPP (SMAZnPP), were found to inhibit cytokine-dependent and spontaneous proliferation in all 3 AML cell lines as well as in primary AML cells. Growth inhibitory effects of SMA-ZnPP and PEG-ZnPP were dose-dependent with IC50 values ranging between 1 and 20 μM, and were accompanied by apoptosis as evidenced by light- and electron microscopy, Tunel assay, and caspase-3 activation. Finally, we were able to demonstrate that SMA-ZnPP inhibits cytokine-dependent proliferation of CD34+/CD38+ and CD34+/CD38- AML progenitor cells in vitro in all patients as well as leukemiainitiation of AML stem cells in NOD-SCID IL-2Rγ(-/-) (NSG) mice in vivo. Together, our data suggest that Hsp32 plays an important role as a survival factor in leukemic stem cells and as a potential new target in AML.     

The generation and antigen-specificity of CD4+CD25+ regulatory T cells

CD4+CD25+ regulatory T cells are essential components of the immune system. They help to maintain immune tolerance by exerting suppressive effects on cells of the adaptive and innate immune system. In the last few years there has been an abundance of papers addressing the suppressive effects of CD4+CD25+ regulatory T cells and their putative role in various experimental disease models and human diseases. Despite the enormous amounts of data on these cells a number of controversial issues still exists. CD4+CD25+ regulatory T cells were originally described as thymus-derived anergic/suppressive T cells. Recent papers however indicate that these cells might also be generated in the periphery. Due to the thymic development of CD4+CD25+ regulatory T cells it was thought that these cells were specific for self-antigens. Indeed it was shown that CD4+CD25+ regulatory T cells could be positively selected upon high affinity interaction with self-antigens. However, evidence is accumulating that these cells might also interact with non-self antigens. Finally, in the literature there is conflicting evidence regarding the role of soluble factors versus cell-contact in the mechanism of suppression. The aim of this review is to summarize the evidence supporting these opposing viewpoints and to combine them into a general model for the origin, function and antigen-specificity of CD4+CD25+ regulatory T cells.     

六种重组细胞因子对人骨髓造血细胞的体外扩增效应

目的 :观察造血生长因子对骨髓CD34 +细胞的扩增效应及扩增细胞的分化特性。方法 :采用重组人白细胞介素 (rhIL) 1 +rhIL 3+rhIL6 +重组人粒细胞集落刺激因子 +重组人粒 巨噬细胞集落刺激因子和重组人干细胞因子对人骨髓单个核细胞 (MNC)进行刺激 ,动态观察了经上述细胞因子作用后MNC的增殖效应及液态扩增培养后粒 巨噬集落形成率 (CFU GM ) ,利用流式细胞技术 (FACS)分析了扩增前后CD34 +细胞及其亚群的动态变化。结果 :经上述 6种细胞因子作用 2 4d ,骨髓MNC总数是原代MNC的 38.33倍 ,液态扩增 1 0d后 ,CFU GM的数量达高峰 ,是原代MNC的 1 9.0 4倍 ,2 4d时CFU GM总数降至 3.94倍 ,且集落明显小于扩增初期。扩增至 6～ 1 0d ,CD34 +细胞总数是原代MNC的 1 36 .40～ 90 .40倍 ,1 9d时降为 6 1 .40倍。结论 :外源性造血生长因子对CD34 +骨髓细胞具有较强的扩增效应 ,选择适宜的扩增时间是保证骨髓移植后尽快重建造血功能的重要环节。     

Experimental attempt to produce mRNA transfected dendritic cells derived from enriched CD34+ blood progenitor cells

It Peripheral blood progenitor enriched CD34+ cells (PBPC) are rather often used as stem cell background in cancer patients following high dose therapy. Keeping in mind that precursor dendritic cells (DCs) originate from haematopoietic progenitor cells, purified CD34+ cells might also serve as starting cells for ex-vivo production of DC. The aim of the present study is to develop a clinical grade procedure for ex-vivo production of DC derived from enriched CD34+ cells. Various concentrations of CD34+ cells were grown in gas-permeable Teflon bags with different serum-free and serum-containing media supplemented with GM-CSF, IL-4, TNF-a, SCF, Flt-3L and INF-a. Serum-free CellGroSCGM medium for 7 days followed by CellGroDC medium in 7 days gave equal results as serum-containing medium. Following incubation, the cultured cells containing immature DCs were concentrated and transfected with tumour mRNA from human prostate cancer cell lines employing a highly efficient electroporation procedure. Thawed transfected DCs were able to elicit primary T-cell responses in vitro against antigens encoded by the prostate cancer mRNA as shown by ELISPOT assay using mock-transfected DCs as control. The results of our study show that frozen enriched CD34+ cells can be an alternative and efficient source for production of DCs for therapeutic purpose.     

自身免疫病患者外周血CD34+细胞动员、采集和纯化研究

目的：探讨自身免疫病患外周血造血干细胞动员、采集和纯化的效果及安全性。方法：对17例确诊自身免疫病患予环磷酰胺＋粒细胞集落刺激因子（G－CSF）动员外周血干细胞，CliniMACS系统对其中11例患外周血单个核细胞进行CD34^ 细胞阳性分选，流式细胞仪检测分选前后CD34^ 细胞纯度及淋巴细胞各亚群。结果：1例动员中并发尿崩症，2例动员失败。11例分选后CD34^ 细胞纯度平均96．1％，回收率70．14％；CD3^ 、CD19^ 、CD14^ 细胞分别去除10^3/kg,10^2/kg,10^3/kg。结论：对自身免疫病患采用环磷酰胺＋G－CSF方案动员外周血干细胞方法可靠，安全性好；外周血干细胞的纯化方法可获得高纯度和高回收率的CD34^ 细胞，并极大限度的去除T、B等细胞。     

Preparation of PEG-grafted immunomagnetoliposomes entrapping citrate stabilized magnetite particles and their application in CD34+ cell sorting

Immunomagnetic systems have been used for positive selection of a cell fraction from a mixture using appropriate surface markers with satisfactory results, as haematopoietic CD34+ cells. This work reports on the development of poly(ethylene glycol)-grafted (PEG) immunoliposomes loaded with citrate-magnetite stabilized particles as the separation vehicles for immunomagnetic separations. The magnetic ferrofluid was encapsulated into PEG-liposomes by the DRV methodology. The magnetoliposomes had a liposomal size of approximately 450 nm and a Fe/lipid molar ratio of 1.52+/-0.26, and were retained in the magnetic field created by the MiniMACS system. Anti-CD34 immunomagnetoliposomes with 100 mAb/vesicle were prepared by coupling the My10 mAb and bound specifically for CD34+ KG-1a cells in culture and in mixtures with CD34-cells (CHO or Jurkat). The magnetic cell sorting was carried out in cell mixtures KG-1a/CHO or KG-1a/Jurkat with different initial% of CD34+ Kg-1a cells. For 10(6) positive cells and 100 microM of immunomagnetoliposomes, the capture efficiency was > 85% and independent of the starting percentage of CD34+ cells. The decrease of the final purity, when the starting percentage of CD34+ cells decreases and, dependent of the CD34- cell line used, point to the degree of non-specific cell binding of My10-immunomagnetoliposomes as being crucial, among of the methodological aspects as the number of starting CD34+ cells. The CD34+ cells isolated retained the viability with an estimated recovery of 45-50%.     

造血移植物中CD3~+CD8~(low)和CD3~+CD4~-CD8~(low)细胞亚群的检测及分析

目的检测造血移植物(正常人骨髓、脐血及动员后外周血)中CD3+CD8low和CD3+CD4-CD8low细胞亚群,探讨其促进造血干/祖细胞植入异基因骨髓的功能,以期拓宽脐血移植的应用范围。方法直接三色免疫荧光标记流式细胞术检测。结果CD3+CD8low和CD3+CD4-CD8low细胞亚群占CD3+细胞亚群的比例在骨髓中最高,为(8.61±1.40)%,动员后外周血次之,为(5.11±0.76)%,脐血最低,为(3.31±0.88)%(P<0.01);"初始"T(CD45RA+CD45RO-)细胞亚群占CD8low细胞亚群的比例为脐血(94.26±2.46),骨髓(58.68±7.57),动员后外周血(73.21±3.60),"初始"T占CD8high细胞亚群的比例为脐血(82.63±3.16),骨髓(38.69±3.24),动员后外周血(51.58±4.23),各移植物中"初始"T细胞亚群占CD8low细胞亚群的比例均高于其占CD8high细胞亚群的比例(P<0.01),CD8low细胞亚群中"初始"T与"记忆"T(CD45RA-CD45RO+)细胞亚群的比例均高于二者在CD8high细胞亚群的比例。结论脐血CD8low和CD8lowCD4-细胞占CD3+细胞的比例明显低于骨髓,可能是脐血移植植入延迟的原因之一;"初始T与"记忆"T细胞亚群的比例增高,可能与CD3+CD8low细胞亚群维持和诱导免疫耐受,不引起移植物抗宿主病有关。     

Stimulation-dependent induction of CD154 on a subset of CD4+ FoxP3+ T-regulatory cells

CD40-ligand/CD154 is predominantly expressed on activated CD4 T cells and plays a central role in regulating CD4 T-cell-dependent responses. To define the relative abilities of CD4 T-cell functional subsets in the induction of CD154--specifically FoxP3- effector, versus FoxP3+ regulatory, CD4 T cells--multiple CD4 T cell preparations were isolated from B6 and B6.FoxP3-GFP mice and stimulated in vitro to examine the kinetics of stimulation-dependent CD154 expression. CD154 was induced in 40-60% of total CD4 T cells in various cell preparations. However, despite similar kinetics of CD154-induced expression, the average percentage of CD154 expression among CD4+ FoxP3+ T regulatory (Treg) cells was only about 4-9%. Such differential, stimulation-dependent CD154 induction by total CD4+ T cells versus CD4+ FoxP3+ Treg cells was consistent, despite multiple stimulation conditions utilizing a variety of cell preparations of different composition. Similar induction of CD154 occurred irrespective of whether the CD4+ FoxP3+ Treg cells were first sorted to 98% purity and stimulated in vitro alone, or stimulated as non-purified cells in the presence of CD4+ FoxP3- T effector cells, suggesting that CD154 induction by CD4+ FoxP3+ Treg cells is regulated by cell-intrinsic mechanisms. Differential CD154 induction may be a key factor in determining the distinguishable functions of FoxP3- T-effector, versus FoxP3+ Treg, CD4+ T cells.     

Head and neck cancer triggers increased IL-6 production of CD34+ stem cells from human cord blood

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) are infiltrated by various kinds of immune cells, which show massively impaired immune functions. The influence of HNSCC on CD34 + progenitor cells from human cord blood was analyzed. MATERIALS AND METHODS: CD34+ cells were isolated from human cord blood by 'magnetic bead separation' using magnetically labelled antibodies. Immunofluorescent staining of CD34+ cells in solid HNSCC was carried out. Cytokine levels of IL-6, IL-8, and IL-10 were analyzed with flow cytometry using the BD CBA Human Soluble Protein Flex Set system (Becton Dickinson). RESULTS: We demonstrated that HNSCC triggered CD34+ cells to produce increased levels of the tumor-promoting cytokine IL-6 and thus they participate in the development of the microenvironment of head and neck cancer. CONCLUSION: HNSCC modulates the cytokine secretion profile of tumor infiltrating cells to escape from efficient immune responses und to trigger its own malignant progression.     

Human CD34+ progenitor hematopoiesis in liquid culture for in vitro assessment of drug-induced myelotoxicity

Utilization of validated CFU-GM assays for myelotoxicity screening is hampered by its labor-intensive and low-throughput nature. Herein, we transformed the defined CFU-GM assay conditions and IC₉₀ endpoint into a higher throughput format. Human CD34⁺ hematopoietic progenitors were cultured in a 96-well plate for 14 days with the same cytokine (rhGM-CSF) used in the CFU-GM assay. Expansion and differentiation toward myeloid lineages were manifested by characteristic changes in nuclear and cytoplasmic morphology and by temporal expression patterns of CD34, CD11b and CD13 markers. Inhibition of CD34⁺ cell myelopoiesis by 12 anticancer drugs known to induce myelotoxicity in the clinic was quantifiable using either general cytotoxicity endpoints (cell growth area or total nucleus count) or lineage specific readouts (count of cells expressing CD11b and/or CD13). The IC₅₀ and IC₉₀ values derived from the concentration-response curves of 14-day drug exposure in CD34⁺ cell culture were highly correlated with those from the international validation study of the CFU-GM assay, demonstrating capability to assess general cytotoxicity, cell proliferation and myelopoiesis simultaneously. These results suggest that this human CD34⁺ hematopoietic progenitor cell assay can be used as a direct replacement for the validated, low throughput CFU-GM assay, and could expand application of in vitro myelotoxicity testing.     

Targeting CD4+CD25+FoxP3+ regulatory T-cells for the augmentation of cancer immunotherapy

CD4+CD25+FoxP3+ T-regulatory (Treg) cells are vital to the maintenance of peripheral self tolerance and are implicated in tolerance to foreign antigens. Increasing evidence shows that Treg cells may also play an important role in immune evasion mechanisms employed by cancer. Treg cells are actively recruited and induced by tumors to block innate and adaptive immune priming, effector function and memory response, which can inhibit the efficacy of therapeutic cancer vaccines. As such, modulation of Treg cell function in cancer has been studied using various approaches, with encouraging preclinical and clinical findings. However, controlled and effective modulation of Treg cell function for cancer therapeutics will be contingent on a better understanding of the molecular basis of Treg cell interaction with tumor cells and ensuing immunosuppressive mechanisms.