【作者】 余佳；

【导师】 张俊武；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 血细胞形成是生物体发育和生命维持最重要的环节之一。机体内对血细胞形成过程的调控包含两方面：(1)造血干细胞的自我更新，即多能造血干细胞自我复制；(2)造血干／祖细胞的分化，即造血干／祖细胞向至少8个血细胞链系的分化决定并继续发育分化为成熟的细胞。造血干细胞向红系的分化决定和继续分化发育成熟是多个基因有序地开启(或表达增强)和关闭(或表达降低)的结果。它们的表达异常可能导致血液病的发生。K562细胞系是一种人红白血病细胞系，基本处于多能髓样祖细胞阶段而停止继续分化。在氯高铁血红素(Hemin)诱导下可继续红系分化过程。为进一步获得分化过程中相关基因表达的信息，我们应用改良的差异显示RT-PCR技术(DDRT-PCR)，分析K562细胞在Hemin诱导的红系分化前后的基因表达变化，共获得357条差异表达带。对其中差异表达明显的314条进行再扩增、克隆和测序，其中201条表达上调，85条表达下调。对这些差异表达EST的生物信息学分析发现，237个EST与GenBank中已知序列有95％以上同源性，45个为只与dbEST有高度同源的序列，4个未发现同源序列。这些已知基因多为信号转导分子、分化相关分子、细胞凋亡相关分子、肿瘤相关分子、染色质结构蛋白和氧化还原酶及其它酶类等，还有一些已知cDNA而未知功能的基因和几个可能的新基因。同时，我们也运用Microarray技术分析了K562细胞红系分化前后的miRNA表达，获得了21个差异表达miRNA。这些结果为确定红系分化相关基因及调控网络提供了重要线索。miRNA是一类长度约为21-23个核苷酸的重要调节分子，它可以通过mRNA剪切或翻译抑制的方式在转录后水平抑制蛋白编码基因表达。迄今为止在植物和动物中已发现编码miRNA的基因1000余个。miRNA在许多发育和生理过程中起着重要的作用，但目前已阐明部分功能和作用机制的miRNA只有少数。近来Bartel等报告mir-223在小鼠骨髓中特异性表达。为研究mir-223在人类造血中的功能作用，本论文首先用Northern杂交方法分析了其在人正常组织中的表达，发现mir-223在人淋巴结、肝和骨髓中高表达，在其他组织中低或无表达，这与Bartel在小鼠组织的杂交结果有部分区别。随后通过密度梯度离心结合抗体磁珠分选的方法从脐血单个核细胞中分离出5种链系细胞(T、B、粒、单核和红系)，检测了mir-223表达，结果表明mir-223在红系和T系表达较低，在B系中等程度表达，而在单核系和粒系中高表达。之后检测了mir-223在9个白血病细胞系中的表达，发现其不但在2个髓系来源白血病细胞系HL-60和THP-1中表达，也在2个红白血病细胞系K562和HEL中表达。但在小鼠红白血病细胞系MEL585和GM979中却未检测出mir-223表达。这些情况暗示，在作为比小鼠更高级的哺乳动物—人中，miRNA可能在发育过程中调控更多的生理和病理进程。为进一步研究mir-223在人红系分化发育中的功能，我们以K562和HEL为研究模型，分析外源性抑制或过表达成熟mir-223后对两种细胞生长和分化的影响。结果表明，外源性抑制或过表达成熟mir-223对两种细胞的增殖和凋亡没有影响，而外源性抑制了成熟mir-223的两种细胞在用Hemin向红系诱导后，相比于对照Hemin诱导组，均表现出一定程度的红系分化增强。这表明mir-223可能在人红系分化中具有一定抑制作用，但尚需进一步研究。许多证据表明多个miRNA可能以协同方式在某一生物过程中发挥作用。本研究首先使用Altuvia等的方法分析了所有已注册的326个人miRNA基因的基因组定位情况，发现148个miRNA基因组织在51个miRNA基因簇里，并用启动子分析得到验证。序列相似性分析揭示在不同的miRNA基因簇之间存在许多高度同源的miRNA基因。为进一步探究这些成簇miRNA在人造血系统中的功能，我们用northern blot方法分析了它们在不同的人白血病细胞系中的表达。结果说明在39个miRNA簇中，属于同一个簇的miRNA成员在不同的人造血细胞系中具有一致性表达。不一致的表达在另12个基因簇中发现。从中鉴定了几个造血链系特异或丰富表达的miRNA簇(例如：mir-29c，mir-302，mir-98，mir-29a和let-7a-1簇等)以及几个链系特异表达的miRNA(例如：mir-181c，mir-181d，mir-191和mir-136等)，提示这些miRNA可能在相应的人造血链系分化或白血病发生过程中起重要作用。这些结果为造血细胞分化机制特别是红系分化详细机制和调控网络的揭示提供了有意义的资料和重要线索，并可能对造血细胞分化相关疾病的研究和治疗提供潜在的线索。更多还原

【Abstract】 Hematopoiesis is one of the most important links in organism development and lifemaintenance. Regulation of hematopoiesis consists of two integral aspects: (1) theself-renewal of hematopoietic stem cells (HSCs)—how pluripotential stem cellsreplicate themselves; (2) the differentiation of hematopoietic stem/progenitor cells—howstem/progenitor cells undergo progressive differentiation decision of at least eightdistinct lineage potential and develop into mature, terminally differentiated cells.Erythropoiesis is a process by which pluripotent hematopoietic stem cells give riseto erythrocytes undergoing a series of differentiation decision and proliferation stages byopening and closing of related genes in proper order or increasing and decreasingexpression of related genes. K562 is an erythroleukemia cell line, which is situated in thecommon progenitor stage of megakaryocytic and erythroid lineages of the hematopoieticstem cell differentiation and its normally following differentiation is blockaded. However,K562 cells can be induced to erythroid differentiation by hemin. To get more informationon gene regulation during erythroid differentiation of K562 cells, we applied modifieddifferential display reverse transcription polymerase chain reaction (DDRT-PCR) methodto identify genes showing differential expression in uninduced and hemin-induced K562cells. We totally obtained 357 differential ESTs. We selected 314 cDNA fragments forre-amplifying, cloning, sequencing and analyzing with bioinformatics methods. Amongthem, 201 were upregulated and 85 downregulated; 237 differential ESTs were found tohave more than 95% homology to known GenBank sequences and 45 represented cDNA sequences with only dbEST database matches and 4 ESTs have no any database matches.These known genes are involved in cell signaling transductors, factors associated cancerand apoptosis, factors associated differentiation, chromatin structure proteins, enzymesassociated redox state, some unknown function cDNAs and unknown genes. At the sametime, we also obtained 21 differential miRNAs with differential expression s inuninduced and hemin-induced K562 cells by miRNA-Microarry analysis. Our resultsprovide vital clues for identifying genes and regulation network related to erythroiddifferentiationMicroRNAs (miRNAs) are a novel class of conserved 21-23 nucleotides long RNAswhich can negatively regulate gene expression at post-trancriptional level by mRNAdegradation and translation repression. So far, one thousand of miRNAs have been foundin animals and plants. They may have diverse biological functions in developmental andphysiological processes, but only a handful of miRNAs have been carefully studied.Recently, mir-223 was reported to be specifically expressed in bone marrow of mice.Here, to study the function of mir-223 in human hematopoiesis, we firstly analyzed theexpression of mir-223 in human normal tissues, and found that its expression was higherin lymphonode, liver and bone marrow, whixh is different to published mouse data byBartel et al. Then we isolated 5 normal hematopoietic lineage cells (T cells, Bcells,granulocytes, monocytes and erythrocytes) from cord blood by density gradientcentrifugation and magnetic cells sorting and detected the expression of mir-223 in them.mir-223 expression was low in T cells and erythrocytes, moderate in B cells and high inmyeloid cells. Subsequently we also detected mir-223 expression in 9 human leukemiacell lines and found that its expression was high not only in HL-60 and THP-1 derivedfrom myeloid cells but also in 2 erythroleukemia cell lines—K562 and HEL. However,we failed to detect its signal in two mouse erythroleukemia cell lines—MEL585 andGM979. These cases indicated that miRNA may posses more biological function inhuman than mouse in diverse developmental and physiological processes. To studypossible functions of mir-223, we examined if expression inhibiting or overexpressing of mature mir-223 could affect growth and differentiation of K562 and HEL cells. Resultsshowed that inhibiting or overexpressing mature mir-223 exogenously did not influenceproliferation and apoptosis of the cells, but inhibiting mature mir-223 exogenously couldincrease the ratio of cells that express globins in hemin-induced HEL and K562 cells.This case implied that human mir-223 meight possess inhibition function for erythroiddifferentiation of K562 and HEL cells. Further studies should be performed todemonstrate this observation.Recently, following the discovery of miRNA gene clusters, some groups indicatedthat miRNAs might work in combination to accomplish their function throughout manybiological processes. In this study, we first analyzed the clustering properties of allregisted 326 human miRNA genes and found that 148 miRNA genes are organized in atotal of 51 clusters. Subsequent promoters analysis demonstrated our results. Alignmentof the miRNA sequences in different clusters revealed a significant number of miRNAparalogs among the clusters, implying an evolution process targeting the potentiallyconserved roles of these molecules. Then we performed northern blot analysis toexamine expression profiling of all clustered miRNAs in several human leukemia celllines. Consistent expression of the miRNAs in a single cluster was revealed in 39 clusters,while inconsistent expression of members in a single cluster was detected in the other 12clusters. Meanwhile, we identified several hematopoietic lineage-specific or -enrichedmiRNA clusters (e.g., the mir-29c, mir-302, mir-98, mir-29a and let-7a-1 clusters) andindividual miRNAs (e.g., mir-181 c, mir-181 d, mir-191 and mir-136). These findings maysuggest vital roles of these miRNA clusters or miRNAs in human hematopoiesis andoncogenesis, and provide clues for better understanding the function and mechanism ofmiRNAs in various biological processes.Our results provide the significant data and important clues for the detailedmechanisms and regulation network of hematopoiesis and especial erythroiddifferentiation, and possibly provide potential clues for studying and curing the diseasesrelated to hematopoietic differentiation.更多还原