造血干细胞(CD34+)表达谱数据分析

造血干细胞分化的潜能及自我更新的分子机制是造血干细胞研究中最重要的领域.通过对来自造血干细胞(CD34+)的EST(Expressed Sequence Tags)和SAGE(Serial Analysis of GeneExpression)数据进行系统生物信息学分析,发现了造血干细胞除表达大量下游分化细胞的分子标志外,还表达一些非造血组织的特异基因.在分子调控方面,反义RNA可能是一种非常重要的调控手段.     

骨髓造血微环境与基质细胞的基因治疗

造血干细胞是所有免疫细胞和血细胞的来源，而造血微环境是造血干细胞增殖、发育和分化的场所，对造血干细胞发育为成熟血细胞起重要的调节作用。造血微环境（HematopoieticMicroenvironment，HME）的概念1970年首先由坦廷（Tentin）提出，当时认为它由骨髓中微血管系统、神经、网状细胞、细胞外基质及纤维组织等构成，对造血干细胞的分化成熟起调节作用。以后的研究证明，微环境中的基质细胞（StrormalCell，SC）及一些造血刺激因子是造血微环境的主要成份。1977年德克斯特（Dexter）建立了体外长期骨髓细胞培养系统地Long-TermBoneM…     

造血干细胞免疫表型特征的研究进展(综述)

造血干细胞（ＨＳＣ）是一群具有高度自我更新和多向分化潜能的造血前体细胞。临床上常采用不同来源的造血干细胞进行输注移植，以治疗各种疾病。另外，由于造血干细胞的特殊性能，它们可作为基因治疗最理想的靶细胞，在治疗遗传性疾病、恶性肿瘤、严重免疫缺陷及感染性疾病等方面具有广阔的应用前景。因此，造血干／祖细胞的分离纯化、增殖分化性能及体外扩增等方面的研究是近年来备受关注的研究课题。现已知造血于细胞是由一群不均一的细胞群体所组成，不同年龄等级的造血干细胞的表面抗原、免疫表型表达各不相同，生物学特征也有一定的差…     

基于布尔网络的造血细胞谱系分化选择分析

造血干细胞通过分化获得不同的成熟血细胞类型以满足不同的生理需求.通过整合两个已有的基因调控网络并进行修正，构建了综合调控网络以及对应的逻辑模型，可以更全面地反映不同基因在造血干细胞分化过程中的作用.该网络的稳态结果包含了两个小网络中涉及的大部分细胞类型，与实际的ChIP-seq数据结果一致.对综合调控网络进行瞬态扰动与持续扰动，分析了6种主要细胞类型之间正常分化所需要的最简扰动策略.获得的整合调控网络与逻辑模型可以为理解造血干细胞的分化特异性选择提供一些定性的参考依据，同时分析得到的扰动策略可以为实际的细胞培养实验提供一些方案上的建议.     

新疆生地所处理油田作业污水研究取得重要进展

[遗传与发育生物学研究所]近年来，诱导多功能干细胞（iPS）引发了生命科学与医学界干细胞研究的热潮。于细胞广泛应用的前提是明确其自我更新和定向分化的调控机制。OCT4是参与调控胚胎干细胞自我更新和维持其全能性的最为重要的转录因子之一，同时也是体外建立诱导多功能干细胞（iPS）的关键基因。     

胚胎干细胞的研究进展

胚胎干细胞（embryonic stem cell,ES细胞）是来源于囊胚内细胞团的一种多能细胞,具有分化的多向性和长期增殖能力,已经广泛用于生命科学的许多领域,它在医学方面的应用也成为医学领域的研究热点。本文综述了胚胎干细胞在诱导分化为神经细胞、造血干细胞、内皮细胞等方面的研究进展及在临床应用前景。     

补髓生血颗粒剂对慢性再生障碍性贫血患者影响的实验研究

应用单标及双标免疫组织化学技术和酶联免疫法,对慢再生障碍性贫血患者骨髓CD34细胞、Fas 细胞、CD34 mP53表达细胞及血清细胞子IL-2、IL-3进行观察,结果表明,补髓生血颗剂能提高细胞、CD34mP53表达细胞百分率(P＜0.05),降低Fas 细胞分率(P＜0.05),降低IL-2水平(P＜0.05).提示:1)补髓生血颗粒剂可能能通过提高抑制凋亡因mP53表达,降低促进凋亡Fas抗原表达抑制血干细胞的过度凋亡.2)补髓生血颗粒剂能够降低造血负调控因子水平,同时提高造血正调控因子水平,并可能通过造血因子调控凋亡相关基因的表达,从而抑制造血干细胞的过度凋亡.     

QIAquick Gel Extraction Kit提取和纯化PCR产物用于AML中GATA-2基因突变的分析

转录因子ＧＡＴＡ家族是１９８９年首次发现的锌指结构家族转录因子。其中ＧＡＴＡ２在造血干细胞的增殖和分化中起重要作用［１］。绝大多数急性髓细胞白血病（ＡＭＬ）细胞表达ＧＡＴＡ－２基因［２］。在分析ＧＡＴＡ－２基因表达中，发现ＧＡＴＡ－２突变的现象［...     

神经元素3蛋白(Ngn3)诱导大鼠骨髓间充质干细胞向胰岛素表达细胞分化的初步研究

胰腺发育相关基因神经元素3（neurogenin3,Ngn3）,属于碱性螺旋-环-螺旋（basichelix-loop-helix,bHLH）家族的转录因子,对胰腺内分泌细胞的发育及分化至关重要,且被认为是胰腺祖细胞的标志蛋白质．之前的研究发现,全长Ngn3蛋白具有蛋白质转导功能,可以高效进入多种真核细胞系．近年来多项研究显示,骨髓间充质干细胞是一种具有多向分化潜能的多能干细胞．利用蛋白质转导技术,将体外表达纯化的胰腺发育重要因子Ngn3蛋白导人体外分离培养的大鼠骨髓间充质干细胞中,诱导其向胰岛素表达细胞分化．结果显示,经Ngn3蛋白诱导的大鼠骨髓间充质干细胞出现细胞聚集生长现象,并有形状类似胰岛的细胞团出现．RT-PCR结果显示经Ngn3蛋白诱导的大鼠骨髓间充质干细胞可以有效表达胰岛素,并且一些β细胞相关基因如胰十二指肠同源盒基因-1（pancreatic-duodenal homeoboxl,Pdx-1）,神经分化因子（neurogenic differentiation,NeuroD）,同源结构域基因4（pairedboxgene4,Pax4）,葡萄糖转运体-2（gluoase transporter2,Glut-2）都有表达．细胞免疫荧光也可检测到胰岛素的表达．通过Ngn3蛋白质诱导分化大鼠骨髓间充质干细胞的初步研究,探索了应用骨髓间充质干细胞体外诱导分化获得胰岛素表达细胞的可行性,为糖尿病治疗提供一定的实验依据．     

全反式维甲酸对脐血红系祖细胞增殖过程中HOXB6基因表达的影响

探讨脐血造血干祖细胞向红系增殖过程中HOXB6 mRNA表达及全反式维甲酸对HOXB6 mRNA表达的影响.采用体外培养技术,以全反式维甲酸持续干扰造血干祖细胞,观察集落生成情况,采用实时荧光定量PCR技术检测造血祖细胞增殖分化过程中HOXB6基因的表达水平,用DNA相对拷贝数和RNA表达相对量表示HOXB6基因相对表达量.结果表明,人类造血干祖细胞向红系祖细胞增殖分化过程中,各祖细胞HOXB6基因均表达,与正常对照组比较,全反式维甲酸可上调HOXB6基因的表达,说明HOXB6可能是造血干祖细胞向红系祖细胞正常增殖分化过程中的调控基因之一.     

柴胡皂苷d对肝星状细胞的作用分析

从传统药物中寻找生物活性成分是开发抗肝纤维化药物的重要途径。人们发现中药柴胡成份柴胡皂苷d（SSd）可以抑制肝星状细胞（HSC）活化，但是相关生物学机制的研究较少。本研究以体外诱导活化的HSC为肝纤维化细胞模型，探究SSd抗肝纤维化效果及其分子机制。结果显示，SSd能够抑制HSC增殖和纤维化相关基因转录，并且可能通过激活细胞凋亡通路和抑制线粒体能量代谢两条途径诱导HSC发生凋亡。本研究表明，SSd有希望成为抗肝纤维化的潜在药物。     

Endothelial and perivascular cells maintain haematopoietic stem cells

Several cell types have been proposed to create niches for haematopoietic stem cells (HSCs). However, the expression patterns of HSC maintenance factors have not been systematically studied and no such factor has been conditionally deleted from any candidate niche cell. Thus, the cellular sources of these factors are undetermined. Stem cell factor (SCF; also known as KITL) is a key niche component that maintains HSCs. Here, using Scf(gfp) knock-in mice, we found that Scf was primarily expressed by perivascular cells throughout the bone marrow. HSC frequency and function were not affected when Scf was conditionally deleted from haematopoietic cells, osteoblasts, nestin-cre- or nestin-creER-expressing cells. However, HSCs were depleted from bone marrow when Scf was deleted from endothelial cells or leptin receptor (Lepr)-expressing perivascular stromal cells. Most HSCs were lost when Scf was deleted from both endothelial and Lepr-expressing perivascular cells. Thus, HSCs reside in a perivascular niche in which multiple cell types express factors that promote HSC maintenance.     

Haematopoietic stem cells do not asymmetrically segregate chromosomes or retain BrdU

Stem cells are proposed to segregate chromosomes asymmetrically during self-renewing divisions so that older ('immortal') DNA strands are retained in daughter stem cells whereas newly synthesized strands segregate to differentiating cells. Stem cells are also proposed to retain DNA labels, such as 5-bromo-2-deoxyuridine (BrdU), either because they segregate chromosomes asymmetrically or because they divide slowly. However, the purity of stem cells among BrdU-label-retaining cells has not been documented in any tissue, and the 'immortal strand hypothesis' has not been tested in a system with definitive stem cell markers. Here we tested these hypotheses in haematopoietic stem cells (HSCs), which can be highly purified using well characterized markers. We administered BrdU to newborn mice, mice treated with cyclophosphamide and granulocyte colony-stimulating factor, and normal adult mice for 4 to 10 days, followed by 70 days without BrdU. In each case, less than 6% of HSCs retained BrdU and less than 0.5% of all BrdU-retaining haematopoietic cells were HSCs, revealing that BrdU has poor specificity and poor sensitivity as an HSC marker. Sequential administration of 5-chloro-2-deoxyuridine and 5-iodo-2-deoxyuridine indicated that all HSCs segregate their chromosomes randomly. Division of individual HSCs in culture revealed no asymmetric segregation of the label. Thus, HSCs cannot be identified on the basis of BrdU-label retention and do not retain older DNA strands during division, indicating that these are not general properties of stem cells.     

Identification of the haematopoietic stem cell niche and control of the niche size

Haematopoietic stem cells (HSCs) are a subset of bone marrow cells that are capable of self-renewal and of forming all types of blood cells (multi-potential). However, the HSC 'niche'--the in vivo regulatory microenvironment where HSCs reside--and the mechanisms involved in controlling the number of adult HSCs remain largely unknown. The bone morphogenetic protein (BMP) signal has an essential role in inducing haematopoietic tissue during embryogenesis. We investigated the roles of the BMP signalling pathway in regulating adult HSC development in vivo by analysing mutant mice with conditional inactivation of BMP receptor type IA (BMPRIA). Here we show that an increase in the number of spindle-shaped N-cadherin+CD45- osteoblastic (SNO) cells correlates with an increase in the number of HSCs. The long-term HSCs are found attached to SNO cells. Two adherens junction molecules, N-cadherin and beta-catenin, are asymmetrically localized between the SNO cells and the long-term HSCs. We conclude that SNO cells lining the bone surface function as a key component of the niche to support HSCs, and that BMP signalling through BMPRIA controls the number of HSCs by regulating niche size.     

小鼠造血干细胞的分离、培养及重组慢病毒载体介导的离体hFⅨ基因表达

造血干细胞是基因治疗理想的靶细胞之一,尤其适用于遗传性血液病.而重组慢病毒载体能高效感染造血干细胞,成为造血干细胞途径基因治疗的理想载体.从小鼠骨髓细胞中分离出单个核细胞(MNCs)进行体外悬浮培养,并用免疫磁珠法分离得到高纯度的小鼠Lin-CD117+造血干细胞(HSCs).体外悬浮培养期间,添加细胞因子的造血干细胞的细胞数和集落数逐渐增加,而未添加细胞因子的对照组的细胞数量无明显增加,细胞集落递减.用磷酸钙介导的共转染法制备了携带FⅨ基因的FUXW重组慢病毒,用慢病毒载体分别感染从ICR小鼠和C57小鼠中分离得到的MNCs,7d后测得细胞上清中hFⅨ的表达量分别为41.7±4.2和34.5±6.6ng/mL,而慢病毒感染C57小鼠造血干细胞,添加细胞因子组上清中hFⅨ的表达量为46.6±5.7ng/mL,不添加细胞因子组为33.3±4.8ng/mL.实验结果表明,重组FUXW慢病毒载体可有效感染小鼠单个核细胞和Lin-CD117+造血干细胞,添加细胞因子可提高转移基因的表达量.     

Inhibitory receptors bind ANGPTLs and support blood stem cells and leukaemia development

How environmental cues regulate adult stem cell and cancer cell activity through surface receptors is poorly understood. Angiopoietin-like proteins (ANGPTLs), a family of seven secreted glycoproteins, are known to support the activity of haematopoietic stem cells (HSCs) in vitro and in vivo. ANGPTLs also have important roles in lipid metabolism, angiogenesis and inflammation, but were considered 'orphan ligands' because no receptors were identified. Here we show that the immune-inhibitory receptor human leukocyte immunoglobulin-like receptor B2 (LILRB2) and its mouse orthologue paired immunoglobulin-like receptor (PIRB) are receptors for several ANGPTLs. LILRB2 and PIRB are expressed on human and mouse HSCs, respectively, and the binding of ANGPTLs to these receptors supported ex vivo expansion of HSCs. In mouse transplantation acute myeloid leukaemia models, a deficiency in intracellular signalling of PIRB resulted in increased differentiation of leukaemia cells, revealing that PIRB supports leukaemia development. Our study indicates an unexpected functional significance of classical immune-inhibitory receptors in maintenance of stemness of normal adult stem cells and in support of cancer development.     

Lkb1 regulates cell cycle and energy metabolism in haematopoietic stem cells

Little is known about metabolic regulation in stem cells and how this modulates tissue regeneration or tumour suppression. We studied the Lkb1 tumour suppressor and its substrate AMP-activated protein kinase (AMPK), kinases that coordinate metabolism with cell growth. Deletion of the Lkb1 (also called Stk11) gene in mice caused increased haematopoietic stem cell (HSC) division, rapid HSC depletion and pancytopenia. HSCs depended more acutely on Lkb1 for cell-cycle regulation and survival than many other haematopoietic cells. HSC depletion did not depend on mTOR activation or oxidative stress. Lkb1-deficient HSCs, but not myeloid progenitors, had reduced mitochondrial membrane potential and ATP levels. HSCs deficient for two catalytic α-subunits of AMPK (AMPK-deficient HSCs) showed similar changes in mitochondrial function but remained able to reconstitute irradiated mice. Lkb1-deficient HSCs, but not AMPK-deficient HSCs, exhibited defects in centrosomes and mitotic spindles in culture, and became aneuploid. Lkb1 is therefore required for HSC maintenance through AMPK-dependent and AMPK-independent mechanisms, revealing differences in metabolic and cell-cycle regulation between HSCs and some other haematopoietic progenitors.     

神经网络模型对造血干细胞体外扩增效果评价及预测

采用神经网络技术对造血干细胞HSCs体外扩增能力建立了评价及预测模型.总结了前人的实验结果,共获得341组数据.选取细胞接种密度、细胞因子组合、细胞来源、血清、基质细胞、反应器类型和培养时问等7个影响因子作为网络输入特征参数,分别对有核细胞(nUClear cells,NCs)、CD34 细胞和成集落细胞(colony-forming units,CFU-Cs)进行体外扩增能力的拟合评价及预测.选取124、90及86组实验数据分别用于NCs、CFU-Cs和CD34 细胞为评价指标的神经网络训练;而17、10及14组实验数据分别用于NCs、CFU-Cs和CD34 细胞为评价指标的神经网络预测.结果表明.对NCs、CFU-Cs和CD34 细胞的区间训练准确率分别为85.5%、86.7%和86.1%;区间预测准确率分别为82.4%、70.0%和71.4%.由此可知.人工神经网络的非线性模拟使定量描述HSCs体外扩增效果与培养条件问的关系及预测HSCs的最佳体外扩增条件成为可能.