HOXB4转录因子在造血干细胞中表达调控机制的研究进展

作为同源盒基因（homeobox gene，hox）家族成员，HOXB4编码一类同源盒DNA依赖的结构域核蛋白，是一类特异性的转录因子，对造血干细胞（hematopoietic stem cells，HSC）自我更新及分化之间的平衡起着重要的调节作用。为此，hoxB4在HSC中的表达调控机制备受关注。相关研究证明，hoxB4的一些上游调控因子，如上游激活因子-1（USF—1）、上游激活因子-2（USF-2）和核因子Y（NF—Y），以及一些造血细胞因子如血小板生成因子（TPO）及Wnt3a信号蛋白等，对hoxB4在HSC中的表达均起着重要调控作用。本文就hoxB4基因的结构、生物学特点及其在HSC中表达的调控机制作一综述。     

脐血CD34+细胞体外扩增时HOXB4基因表达的变化

同源盒基因家族成员HOXIM反映原始造血干／祖细胞（PHSC／PHPC）的自我更新和增殖能力。本研究采用实时定量RT—PCR的方法在mRNA水平上检测HOXIM基因的表达，以观察体外扩增脐血CD34^＋细胞自我更新的水平。结果显示：随着体外培养时间的延长，虽然CD34^＋细胞数增加，但HOXB4表达下降；周后，HOXB4几乎检测不到，与成熟外周血淋巴细胞表达HOXIM的水平相同；CD34^＋细胞与骨髓间充质干细胞（BM—MSC）共培养可以减缓CD34^＋细胞HOXIM表达的下降。结论：脐血CD34^＋细胞体外扩增过程中自我更新能力逐渐下降。CD34^＋与BM—MSC共培养有助于减缓体外扩增的CD34^＋细胞自我更新能力的丧失。     

HOXB家族基因与造血干/祖细胞的功能

最近许多研究证明，同源盒基因(homeobox gene，HOX)家族在正常的造血过程中起着重要的作用：作为一类转录因子，HOX基因产物通过结合到特异的DNA序列来调控靶基因的表达。HOX家族成员之一——HOXB4尤其是HOXB4引起了人们的极大兴趣。研究发现，HOXB4的表达与造血干细胞的自我更新和祖细胞高效增殖功能密切相关，本文就有关这方面的一些新的研究进展作一综述。     

HOXB4基因在造血干细胞自我更新中的调控作用

造血干细胞（hematopoietic stem cell,HSC）具有高度自我更新能力和多向分化潜能,HSC的自我更新是由促进生长的正调控信号和导致凋亡的负调控信号之间的平衡来调控的。在正调控信号中,HOXB4通过激活不同的信号通路增强HSC的自我更新,同时又不影响维持正常稳态造血的调控机制。提高HOXB4的表达水平,能够极大地增强HSC的自我更新功能,但基本不影响细胞的分化、系特异性及终末细胞的形态和功能。不仅如此,HOXB4还可增强胚胎干细胞（embryonic stem cell,ESC）的造血潜能,促进ESC向造血细胞分化。因此,HOXB4和（或）HOXB4的靶基因的表达上调可能在干细胞移植和基因治疗等方面具有广阔的应用前景。本文就HOXB4基因参与调控造血干细胞的自我更新,HOXB4对HSC的分化特异性及终末分化的“零”效应及HOXB4调控HSC自我更新的分子机制等进行综述。     

脐血CD34＋细胞和外周血单个核细胞中HOXB4基因表达及其启动子区CpG岛甲基化状态的研究

本研究旨在通过检测脐血CD34＋细胞和正常人外周血单个核细胞（PBMNC）中HOXB4基因表达水平及其启动子区CpG岛的甲基化水平,初步探讨造血系统中HOXB4基因的表达水平与其启动子区甲基化的相关性。采用半定量RT—PCR检测脐血CD34＋细胞和PBMNC中HOXB4基因的表达,应用亚硫酸氢盐测序法检测这两种细胞中HOXB4基因启动子区CpG岛的甲基化位点。结果发现,CD34＋细胞高表达HOXB4,HOXB4基因启动子区CpG岛未发生甲基化;PBMNc不表达HOXB4,HOXB4基因启动子区在ATG上游-129bp的C碱基发生甲基化。结论：HOXB4基因启动子甲基化程度是其基因表达水平的负性调节机制之一。HOXIM在CD34＋细胞中的高表达与其基因启动子低甲基化有关,而HOXB4在PBMNC中的基因沉默则可能与其启动子区的甲基化密切相关。初步筛选出的HOXB4基因启动子区CpG岛甲基化位点,为后续研究奠定了基础,也为扩增造血祖细胞提供一条新途径。     

Self-renewal of multipotent long-term repopulating hematopoietic stem cells is negatively regulated by Fas and tumor necrosis factor receptor activation

Multipotent self-renewing hematopoietic stem cells (HSCs) are responsible for reconstitution of all blood cell lineages. Whereas growth stimulatory cytokines have been demonstrated to promote HSC self-renewal, the potential role of negative regulators remains elusive. Receptors for tumor necrosis factor (TNF) and Fas ligand have been implicated as regulators of steady-state hematopoiesis, and if overexpressed mediate bone marrow failure. However, it has been proposed that hematopoietic progenitors rather than stem cells might be targeted by Fas activation. Here, murine Lin(-)Sca1(+)c-kit(+) stem cells revealed little or no constitutive expression of Fas and failed to respond to an agonistic anti-Fas antibody. However, if induced to undergo self-renewal in the presence of TNF-alpha, the entire short and long-term repopulating HSC pool acquired Fas expression at high levels and concomitant activation of Fas suppressed in vitro growth of Lin(-)Sca1(+)c-kit(+) cells cultured at the single cell level. Moreover, Lin(-)Sca1(+)c-kit(+) stem cells undergoing self-renewal divisions in vitro were severely and irreversibly compromised in their short- and long-term multilineage reconstituting ability if activated by TNF-alpha or through Fas, providing the first evidence for negative regulators of HSC self-renewal.     

Tet-on调控的人HOXB4慢病毒载体构建

目的构建Tet-on调控的人HOXB4慢病毒载体,为体外扩增造血干细胞并保持其干细胞特性提供实验材料基础。方法应用PCR技术从质粒TAT-HA-HOXB4-Full中扩增编码人HOXB4的cDNA,用限制性内切酶EcoRI分别酶切HOXB4基因片段和带有Tet-on开关的可调控慢病毒载体,经T4DNA连接酶连接获得慢病毒载体Teto-Fuw-hHOXB4。对构建的HOXB4慢病毒载体进行酶切片段凝胶电泳及DNA测序分析。结果酶切片段电泳分析和DNA测序分析证明Tet-on调控的人HOXB4慢病毒载体构建成功。结论正确构建了Tet-on调控的人HOXB4慢病毒载体,为后续的体外扩增造血干细胞及相关研究提供材料。     

造血干细胞自我更新的相关信号分子:作用及途径

背景:造血干细胞数目少,且在体外容易分化,这对其应用于移植存在很大的困难.目的:文章集中阐述了Wnt、Notch、Bmi_1、Shh、HOXB4信号分子在维持造血干细胞自我更新调控中的作用及其途径.方法:以HSC、Wnt、Notch、Bmi_1、Shh、HOXB4为检索词,检索PubMed数据库(2002-01/2008-12).文献检索语种限制为英文.纳入与造血干细胞自我更新相关信号分子密切相关的文献.排除重复性研究和Meta分析.结果与结论:计算机初步检索到216篇文献,对其中30篇文献进行研究.造血干细胞是具有自我更新、较强分化发育和再生能力、可以产生各种类型血细胞的始祖细胞,被广泛应用于治疗血液系统疾病,但是造血干细胞在体外容易分化,这对其应用于移植存在很大的困难.如何使造血干细胞在体外扩增和处理的同时保持造血干细胞的自我更新特性成为关键问题.近年来通过不同信号通路增强造血干细胞自我更新能力的信号分子成为研究热点.文章集中阐述了Wnt、Notch、Bmi_1、Shh、HOXB4在维持造血干细胞自我更新调控中的作用及其途径,发现上述5种信号分子均具有增强造血干细胞自我更新的功能.此外还有一些因素在维持造血干细胞自我更新的过程中起重要作用,如作为内源性因素的一系列转录因子Oct-4、Ehox、Nanog、SCL、Runx1等,探讨它们相互作用形成的调控网络如何调控造血干细胞的自我更新,将成为造血干细胞自我更新领域研究的一个重点.     

Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression

Despite the need for alternative sources of human hematopoietic stem cells (HSCs), the functional capacity of hematopoietic cells generated from human embryonic stem cells (hESCs) has yet to be evaluated and compared with adult sources. Here, we report that somatic and hESC-derived hematopoietic cells have similar phenotype and in vitro clonogenic progenitor activity. However, in contrast with somatic cells, hESC-derived hematopoietic cells failed to reconstitute intravenously transplanted recipient mice because of cellular aggregation causing fatal emboli formation. Direct femoral injection allowed recipient survival and resulted in multilineage hematopoietic repopulation, providing direct evidence of HSC function. However, hESC-derived HSCs had limited proliferative and migratory capacity compared with somatic HSCs that correlated with a distinct gene expression pattern of hESC-derived hematopoietic cells that included homeobox (HOX) A and B gene clusters. Ectopic expression of HOXB4 had no effect on repopulating capacity of hESC-derived cells. We suggest that limitations in the ability of hESC-derived HSCs to activate a molecular program similar to somatic HSCs may contribute to their atypical in vivo behavior. Our study demonstrates that HSCs can be derived from hESCs and provides an in vivo system and molecular foundation to evaluate strategies for the generation of clinically transplantable HSC from hESC lines.     

新型人转录因子hBKLF对K562细胞增殖、分化及血红蛋白合成作用的研究

孕22周的胎肝是胎儿的主要造血器官,其中含有大量与造血相关的调控因子。人碱性Krüppel样因子(humanbasicKrüppel-likefactor,hBKLF)是本研究室从这一时期胎肝cDNA文库中新克隆的转录因子。对其C端的结构分析发现,它含有3个特征性C2H2锌指结构,因此属于KLF转录因子家族。前期的表达谱研究工作显示,hBKLF在成人外周血白细胞、骨髓和胎肝中表达量高,在红系中有表达,这提示它可能与造血相关。本研究以K562细胞为模型,研究hBKLF与造血细胞增殖、分化及血红蛋白合成的关系。构建hBKLF正义及反义真核表达载体,转染K562细胞,经G418筛选4周,得到稳定细胞株;用RT-PCR、Westernblot方法测定转染后K562细胞hBKLF表达水平的变化;以转染空质粒的K562细胞为对照组,在显微镜下直接观察转染正义质粒和反义质粒的两组细胞形态的改变;用MTT法比较增殖速率的变化;用甲基纤维素半固体培养法比较集落形成率的差别;用苯息丁法观察氯高铁血红素(hemin)诱导K562细胞分化后血红蛋白合成水平的差异。结果表明正义质粒和反义质粒经NcoI酶切鉴定构建正确。转染正义质粒的K562细胞(正义组)中hBKLF的mRNA及蛋白质表达水平增加,转染反义质粒的K562细胞(反义组)中hBKLF的mRNA水平降低。增殖活性在反义组、正义组、对照组分别为1.335±0.022、1.067±0.010、1.118±0.014,反义组K562细胞的增殖速率加快,正义组细胞增殖速率减慢(P<0.001)。细胞形态在各组间无明显区别。集落形成率在反义组、正义组、对照组分别为148±13、136±10、141±10,各组间无显著差异(P>0.05)。3组细胞血红蛋白合成水平在hemin诱导后均升高,诱导后血红蛋白升高倍数在对照组、反义组、正义组分别为9.064±1.637、14.360±2.185、4.820±0.404,反义组K562细胞合成血红蛋白能力增加,正义组合成血红蛋白能力下降(P<0.05)。结论hBKLF可以抑制K562细胞的增殖,对血红蛋白合成有负调控作用,但未观察到hBKLF对细胞形态及克隆形成率的影响     

microRNA在人脐血CD34^＋CD38^-、CD34^＋CD38^＋细胞中的差异性表达

为进一步探讨microRNA（miRNA）在造血调控中的作用奠定基础,比较了miRNA在人脐血CD34^＋CD38^-、CD34^＋CD38^＋细胞中的差异性表达。从人脐血中分离单个核细胞（MNC）,应用FACSVantage分选流式细胞仪分选CD34^＋CD38^-、CD34^＋CD38^＋细胞;抽提miRNA后与miRNA芯片杂交,应用生物信息学技术分析miRNA芯片的表达结果。结果发现,miRNA在人脐血CD34^＋CD38^＋细胞的表达水平比在CD34^＋CD38^-细胞的表达水平降低至1／2以下者共11个,表达水平升高至2倍以上者共73个,以上84个miRNA被称为“干细胞性”miRNA。经比较Georgantas等和芯片表达结果,发现有12个（14、29％,12／84）相同的miRNA。部分miRNA经历了类似于CD34在造血细胞表面的发展历程。应用生物信息学技术可寻找到新的与造血调控相关的miRNA簇及miRNA的下游靶基因。结论：“干细胞性”miRNA在正常造血中发挥着重要作用,即miRNA的系统表达模式一造血干／祖细胞基因表达谱一造血干／祖细胞的自我更新和系列定向分化。     

人同源盒基因HOXB4慢病毒载体的构建及其在人脐带间充质干细胞中的表达(英文)

本研究构建人同源盒基因(homeobox gene)HOXB4的慢病毒载体,探讨慢病毒介导的HOXB4感染人脐带间充质干细胞后的变化。利用PCR扩增获得HOXB4,并克隆入慢病毒穿梭载体lenti-shuttle;运用4质粒慢病毒包装系统转染HEK293T细胞,48 h收获慢病毒lenti-HOXB4,并测定慢病毒滴度;将lenti-HOXB4感染人脐带间充质干细胞,在倒置荧光显微镜下观察细胞感染效果,确定最佳的病毒感染复数(MOI);同时,利用RT-PCR、免疫荧光染色、CCK-8、流式细胞术检测HOXB4的表达情况及对细胞生长的影响。结果表明:利用四质粒共转染293T细胞,成功获得lenti-HOXB4,测定的病毒滴度为3×108TU/ml;当MOI为20时,lenti-HOXB4对人脐带间充质干细胞具有较高的转染效率,达80%以上;感染lenti-HOXB4的人脐带间充质干细胞,在mRNA和蛋白水平上均检测到了目的基因的表达,其能促进脐带间充质干细胞的增殖;流式细胞术检测结果显示,HOXB4基因并未明显影响间充质干细胞的表面标志。结论:成功获得带有HOXB4基因的慢病毒并实现在脐带间充质干细胞中表达,HOXB4基因能促进脐带间充质干细胞的大量增殖,并未明显影响脐带间充质干细胞表面标志的表达。     

Recombinant factor VIII expression in hematopoietic cells following lentiviral transduction

Autologous transplantation of gene-modified hematopoietic stem cells may provide a therapeutic strategy for several monogeneic disorders. In previous studies, retroviral gene transfer of coagulation factor VIII (FVIII) into FVIII(-/-) mouse bone marrow (BM) cells did not result in detectable plasma FVIII levels. However, specific immune tolerance was achieved against neo-antigenic FVIII. Here, we used lentiviral vectors to study the ability of various hematopoietic cell types to synthesize and secrete recombinant FVIII. Several myeloid, monocytic and megakaryocytic cell lines (K-562, TF-1, Monomac-1, Mutz-3, Meg-01) expressed FVIII at 2-12 mU/10(4) cells. In contrast, two lymphatic cell lines, BV-173 and Molt-4, were less-efficiently transduced and did not express detectable FVIII. Similarly, peripheral blood-derived primary monocytes were transduced efficiently and expressed up to 20 mU/10(4) cells, whereas primary lymphocytes did not express FVIII. Although human and canine CD34(+) cells were transduced efficiently, the cells expressed very low levels of FVIII (up to 0.8 mU/10(4) cells). Following xenotransplantation of transduced CD34(+) into NOD/SCID mice, ELISA failed to detect FVIII in the plasma of engrafted mice. However, NOD/SCID repopulating cell (SRC)-derived human monocytes isolated from BM of these mice secreted functional recombinant FVIII after culture ex vivo. Again, SRC-derived human lymphocytes did not secrete FVIII. Therefore, certain hematopoietic cell types are able to synthesize and secrete functional recombinant FVIII. Our results show for the first time that transplantation of transduced CD34(+) progenitors may give rise to differentiated hematopoietic cells secreting a nonhematopoietic recombinant protein.     

恶性血液病细胞中tankyrase表达与端粒酶活性关系研究

为研究以白血病为主的恶性血液病细胞中端粒酶活性正向调控基因tankyrase的表达与端粒酶活性的关系并初步探讨tankyrase对端粒酶活性调控的机理和意义 ,以实时定量RT PCR技术对髓细胞系白血病细胞株K5 6 2 ,HL 6 0 ,U937,NB4 ,THP 1,HEL ,Dami,T淋巴细胞性白血病细胞株 6T CEM ,Jurkat和B细胞淋巴瘤细胞株Raji中tankyrase的表达进行检测 ,同时检测端粒酶逆转录酶hTERT的表达确定端粒酶活性 ,并以经磁珠分离的正常人CD3 ,CD19 和CD33 细胞和 10份正常人骨髓单个核细胞做对照。结果发现 :tankyrase在恶性血液病细胞株中的表达明显高于正常对照 (U =19,P <0 .0 1) ,其中髓系白血病细胞株中的表达高于正常人CD33 细胞 ,T淋巴细胞性白血病细胞株和B细胞淋巴瘤细胞株中的表达分别高于正常人CD3 和CD19 细胞。髓系恶性血液病细胞株tankyrase的表达 (0 .0 0 32± 0 .0 0 10 )明显低于淋系恶性血液病细胞株的表达 (0 .0 12± 0 .0 0 16 ) (F =2 3,P <0 .0 1)。Tankyrase与hTERT的表达呈正相关 (相关系数为 0 .395 ,P <0 .0 5 )。结论 :tankyrase在恶性血液病细胞株中呈高表达 ,与端粒酶活性呈正相关 ,提示tankyrase可能是恶性血液病中端粒酶活性增高的原因之一。     

In vitro self-renewal division of hematopoietic stem cells

Little is known about how hematopoietic stem cells (HSCs) self-renew. We studied the regeneration of HSCs in culture. Effects of various cytokines on cell division of CD34(-/low) c-Kit(+)Sca-1(+) lineage marker-negative (CD34(-)KSL) bone marrow cells of the mouse were first evaluated in serum-free single cell culture. We then performed a competitive repopulation assay on divided cells to ask if such cell division involved self-renewal of HSCs.In the presence of stem cell factor (SCF), thrombopoietin (TPO) induced a first cell division of CD34(-)KSL cells more efficiently than did interleukin (IL)-3 or IL-6. Multilineage repopulating cells were detected in a significant proportion of cells derived from single cells in culture with TPO and SCF, although this culture condition led to a substantial decrease in HSC number. These regenerated repopulating cells could be further transplanted into secondary recipients. When paired daughter cells were separately studied, one of a pair gave rise to repopulating cells with self-renewal potential, suggesting asymmetric self-renewal division. This study provides evidence that one HSC regenerates at least one HSC in culture.