【作者】 唐华容；

【导师】 陈方平；

【作者基本信息】 中南大学 ， 内科学， 2009， 博士

【摘要】 第一部分脐带血来源的CD34~+CD38~-细胞的体外纯化和向各系的诱导分化目的:建立一个可行的从脐带血中分选出CD34~+CD38~-细胞的方法,并在体外摸索出有效的粒系、红系和巨核系的分化体系,为后续实验提供可靠的细胞标本。方法:①采用免疫磁珠分选法(magnetic activated cell sorting,MACS)正性分选出CD34~+细胞,再通过二次负性分选选出CD34~+CD38~-的细胞并用流式细胞术检测其纯度和用台盼蓝拒染法检测细胞活率。②在体外应用SCF+IL-3+G-CSF或EPO或TPO细胞因子的组合分别诱导CD34~+CD38~-的细胞向粒系、红系以及巨核系分化。用细胞计数法绘制其各系细胞的增殖曲线及用流式细胞术检测诱导分化的效率。结果:①用抗CD34磁珠第一次分选CD34~+细胞后,CD34~+细胞的纯度可达95.24±1.03%;第二次分选后,CD34~+/CD38~-细胞的纯度为90.23±2.52%。分选前后细胞活力为均可达99%以上。②体外诱导分化14天时,粒系细胞数增加了1186.67±106.1倍,红系细胞数增加了894.67±48.22倍,巨核系细胞数增加了627±49.65倍。③流式细胞术检测的结果表明,在诱导分化的第14天,CD15~+细胞的比率为91.49%,CD235a~+细胞的比率为95.55%,CD41a~+细胞的比率为86.52%。结论:我们建立了有效的MACS分选方法和体外诱导方法,这为我们后续的实验提供了可靠的细胞标本来源。第二部分微小染色质免疫共沉淀方法的建立目的:染色质免疫共沉淀(chromatin immunoprecipitation assay,ChIP)是目前研究蛋白质与DNA相互作用的强有力的技术之一。然而目前的ChIP实验方法最大缺陷要求大量的细胞数,而我们分选的细胞很难达到这个要求。因此我们的目的是建立一种能在少量细胞中进行的ChIP实验方法,称为微小染色质免疫共沉淀(miniChIP)。方法:综合国外相关文献,在传统ChIP方法的基础上建立miniChIP实验方法。并通过运用传统的ChIP实验方法和在此基础建立的miniChIP实验方法对诱导前后的MEL细胞中表达的β珠蛋白基因的不同位点的组蛋白4的乙酰化(acH4)水平进行研究,证实新建的miniChIP实验法方法的可靠性和特异性。β珠蛋白基因的不同位点包括高敏位点2(HS2)、βmaj基因启动子区和Ey基因启动子区。结果:在未处理的MEL细胞中,用miniChIP实验方法观察到HS2和βmaj基因启动子区域存在一定的acH4水平,而Ey基因的启动子区域则检测到极低水平的acH4水平。MEL细胞经过诱导后,HS2位点和βmaj基因启动子区域的acH4水平大大增加,分别增加了2.87和2.26倍。Ey基因的启动子区acH4水平几乎没有变化。这与我们用传统ChIP实验方法观察到的实验结果一致,也与以前别的研究者用传统ChIP实验方法得出的结果相吻合。结论:在传统ChIP实验方法的基础上建立了一种可以在少量的细胞中进行的ChIP实验方法称miniChIP。并在诱导前后的MEL细胞中对此方法进行了验证,证实了miniChIP方法的可行性和可靠性。第三部分造血干细胞髓系分化过程中相关基因的组蛋白修饰特征目的:观察造血分化相关的转录因子和基因在CD34~+CD38~-细胞中以及分化后细胞中的组蛋白修饰特征,探讨染色质构象在造血干细胞多潜能性特性维持和系特异分化中的可能作用。方法:①采用qRT-PCR的方法检测了造血分化相关转录因子和基因在不同类型细胞中的mRNA表达水平。不同类型细胞包括CD34~+CD38~-细胞和诱导分化后的CD15~+细胞、CD235a~+细胞、CD41a~+细胞。造血分化相关转录因子和基因包括早期造血相关转录因子HOXA9;粒系分化相关转录因子PU.1,粒系特异基因MPO、CD11b;红系巨核系分化相关转录因子GATA-1、红系特异基因EPOR和巨核系特异基因CD41a:淋系分化相关转录因子GATA-3、PAX5,淋系特异基因CD3、CD79a。②我们用miniChIP-qPCR实验方法在不同类型细胞中观察并比较了造血分化相关转录因子和基因的启动子区的6种组蛋白修饰的变化。这6种不同的组蛋白修饰分别为活化性组蛋白修饰包括组蛋白3的乙酰化(acH3)、组蛋白4的乙酰化(acH4)、组蛋白3第4位赖氨酸的二甲基化(H3K4me2)、组蛋白3第4位赖氨酸的三甲基化(H3K4me3)和抑制性组蛋白修饰包括组蛋白3第9位赖氨酸的三甲基化(H3K9me3)、组蛋白3第27位赖氨酸的三甲基化(H3K27me3)。结果:①在CD34~+CD38~-细胞中各系分化相关转录因子和基因存在低水平的mRNA表达或不表达,而与早期造血相关的转录因子HOXA9显示高表达。当CD34~+CD38~-细胞向粒系特异分化后,粒系相关基因PU.1、MPO、CD11b表达明显增加;CD34~+CD38~-细胞向红系特异分化后红系相关基因GATA-1、EPOR表达明显增加;CD34~+CD38~-细胞向巨核系特异分化后巨核系相关基因GATA-1、CD41a表达显著增加。同时CD34~+CD38~-细胞系特异分化后非系相关基因未能检测到,以及HOXA9基因的表达显著下降;②在CD34~+CD38~-细胞中各系分化相关转录因子和基因都有一定水平的acH4的修饰和稍低水平的acH3的修饰以及高水平的H3K4me2的修饰,但H3K4me3修饰水平很低。③在CD34~+CD38~-细胞中各系分化相关转录因子和基因都具有低水平H3K9me3和H3K27me3的修饰;④随着CD34~+CD38~-细胞系特异分化后,该系特异基因的acH3、acH4和H3K4me2水平略为增加,但H3K4me3的水平明显增加,同时H3K9me3和H3K27me3修饰仍维持在低水平。非系特异基因的acH3和acH4修饰水平降低,H3K4me3修饰仍维持在低水平,同时有H3K9me3或/和H3K27me3水平的显著增加;⑤在CD34~+CD38~-细胞向终末细胞分化后,与早期造血相关的转录因子HOXA9的启动子区上活化性组蛋白修饰包括acH3、acH4、H3K4me2、H3K4me3显著降低,同时抑制性组蛋白修饰包括H3K9me3和H3K27me3明显增加。结论:①在富含造血干细胞的CD34+CD38-细胞中各系分化相关基因具有一定水平的H3、H4的乙酰化修饰、高水平的H3K4me2修饰以及低水平的H3K4me3修饰和低水平的组蛋白抑制性修饰。这些基因表现为低水平表达或者检测不到表达。②当CD34+CD38-细胞系特异分化后,系相关基因的acH3、acH4、H3K4me2修饰水平维持不变或者略微增加,但H3K4me3修饰水平明显增加,同时保持低水平的抑制性组蛋白修饰,基因表现为高转录状态,而非系相关基因的启动子区上acH3、acH4的修饰水平降低,H3K4me3水平任然维持在低水平状态,但富集了高水平的抑制性的组蛋白修饰标志,基因表现为沉默。③当CD34+CD38-细胞向终末细胞分化后,与早期造血相关的基因HOXA9的启动子区上H3K4me3的修饰水平降低,但抑制性组蛋白修饰标志显著增加,表现为基因表达的沉默。更多还原

【Abstract】 BackgroudHematopoietic stem cells(HSCs) are multipotent stem cells capable of self-renewal and multi-lineage differentiation.HSCs can differentiate to all types of hemapoietic cell lineages including erthryocytes, granulocytes,megakarocytes and T,B lymphocytes.When HSCs give rise to all the blood cell types,there was activation of lineage-specific genes and non-lineage specific genes accompanied by loss of multipotentiation.However,detailed mechanisms behind it are still unclear.Epigenetics research studies regulation of gene expression due to changes of genetic modifications which including DNA methylation, histone modification,genomic imprinting and RNA interference,while no DNA sequence changing.Modifications including DNA methylation, histone acetylation and histone methylation,which are closely related to euchromatin and heterochromatin formation,were studied most recently. Currently,it was shown that epigenetics are of great important in maintaining pluripotency of embryonic stem(ES) cells and can regulate lineage differentiation of ES cells by forming "bivalent" domains.But how it works in HSCs is still unclear.In this study,through investigating histone modifications,which are closely relevant to the chromatin formation,in different cell subgroups,like CD34~+CD38~- cells and differentiatied cells,we try to reveal possible mechanisms of how chromatin structures would maintain HSCs’ multipotent characteristics and regulate relevant genes expression during differentiating,thus providing new perspectives in hemapoiesis,expansion in vitro and transplantation of HSCs,also mechanisms of leukemia generation.This study includes following three parts:Part 1 Sorting of CD34~+CD38~- cells from umbilical cord blood and inducing differentiation in vitroObjective:To establish a feasible method to sort CD34~+CD38~- cells from umbilical cord blood,and stabilize induction system for granulocytes,erythrocytes and megakaryocytes differentiation in vitro, thus providing cell samples for next step experiments.Methods:①Using MACS to positively sort CD34~+ cells,followed by negatively sorting of CD34~+CD38~- cells.FACS and trypan blue methods were used to determine the purity and survive rate of cells.②Using SCF+IL-3+G-CSF or EPO or TPO cytokines to induce CD34~+CD38~- cells differentiate into granulocytes,erythrocytes and megakaryocytes.Cell growth curve was drawn and FACS was adopted to determine the differentiation efficiency.Results:By using CD34 magnetic beads to sort CD34~+ cells,the purity reached up to 95.24±1.03%;After second round sorting,ratio of CD34~+/CD38~- was 90.23±2.52%.The cell survival rate reached up to 99%either before or after sorting.②After induced-differentiation in vitro for 14 days,the number of granulocytes increased up to 1186.67±106.01 folds,erythrocytes increased up to 894.67±48.22 folds and megakaryocytes increased up to 627±49.65 folds.③FACS data show that after induction for 14 days,the ratio of CD15~+ positive cells was 91.49%,CD235a~+ cells was 95.55%and CD41a~+ cell was 86.52%.Conclusion:Efficient MACS sorting and induced-system were established,which provide a reliable source of cells for further experiments.Part 2 Establishment of mini-Chromatin Immunoprecipitation (miniChIP) MethodObjective:ChIP is the most powerful tool currently used in protein-DNA interaction research.However,the drawback of traditional ChIP method is that it needs tremendous number of cells.To collect huge number of primary cells from umbilical cord blood is obviously not easy. Thus,our aim is to establish a ChIP method which requires less cells.Method:Based on the literatures,we established miniChIP method by improving conventional ChIP method.Traditional ChIP method and miniChIP method were both used to compare their efficiency in detecting histone H4 acetylation level inβglobin genes in MEL cells.The feasibility and reliability of miniChIP method was confirmed.Differentβglobin gene loci include HS2,promoter region ofβmaj and Ey.Results:By using miniChIP method,in untreated MEL cells a certain level of acH4 in promoter region of HS2 andβmaj can be detected, but acH4 level in the promoter region of Ey gene was even lower.After MEL cells were induced,the acH4 level in promoter region of HS2 andβmaj gene increased to 2.73 and 2.27 folds,respectively.The acH4 level in Ey gene promoter was almost unchanged.This result was consistent with traditional ChIP method,also as reported before.Conclusion:The traditional ChIP method was improved,which can only use little quantity of cells as sample source.By validating in improved ChIP method,we confirmed its feasibility and reliability.Chapter 3 Characterization of histone modifications during differentiation of HSCs to myeloid cellsObjective:To observe the histone modifications of related transcription factors and lineage specific genes in CD34~+CD38~- cells and commitment cells.Understanding the role of chromatin structure played in maintaining HSCs’ multipotent characterization and differentiation preferences.Method:①Quantitive PCR was adopted to detect the mRNA expression levels of relevant transcription factors and lineage specific genes in CD34+CD38~- cells,CD15~+ cells,CD235a~+ cells and CD41a~+ cells.Transcription factors and genes include early hematopoiesis-related transcription factor HOXA9;granulocyte transcription factor PU.1, granulocyte specific gene MPO and CD11b;erythrocyte/megakaryocyte transcription factor GATA-1,erythrocyte specific gene EPOR and megakaryocyte specific gene CD41a;lymphocyte transcription factor GATA-3 and PAX5,lymphocyte specific gene CD3,CD79a.②miniChIP-qPCR was used to detect six types of histone medications in hematopoiesis relevant transcription factors and lineage specific genes promoter regions,including active modifications acH4,H3K4me2, H3K4me3 and repressive modifications H3K9me3 and H3K27me3.Results:①Differentiating-related transcription factors and lineage specific genes had very low level of expression in CD34~+CD38~- cells while HOXA9,which is relevant to early stage of hemapoiesis,had high mRNA expression;After CD34~+CD38~- cells differentiated,the expression of lineage specific genes increased significantly,while non-lineage specific genes expression can’t be detected,at the same time,the expression of HOAX9 decreased drastically.②A certain level of H4 acetylation and to a lesser extent H3 acetylation together with high level of H3K4me2 and low level of H3K4me3 were present in lineage specific genes in CD34~+CD38~- cells.③Lineage specific genes have low level of H3K9me3 and H3K27me3 modifications in CD34~+CD38~- cells.④As CD34~+CD38~- differentiated,the modifications of acH3,acH4 and H3K4me2 level of lineage specific genes have a little increased,while H3K4me3 level increased greatly.At the same time,H3K9me3 and H3K27me3 modifications maintained at original low level.In non-lineage specific genes,the acH3 and acH4 levels decreased,and H3K4me3 level maintained at low level,at the same time H3K9me3 and H3K27me3 levels increased.⑤After CD34~+CD38~- cells commited,the promoter region of HOXA9,which is relevant to early stage of hematopoiesis,showed decreased active histone modifications including acH3,acH4,H3K4me2 and H3K4me2,while increased repressive histone modifications including H3K9me3 and H3K27me3.Conclusion:①Certain degree of acH3,acH4 and high level of H3K4me2 together with low level of H3K4me3 and low extent of repressive histone modifications(H3K9me3 and H3K27me3) were observed at the promoter of lineage specific genes in HSC-riched CD34~+CD38~- cell population.The expression of these genes shows low or undetected.②After CD34~+CD38~- cells differentiated,active histone modifications of lineage specific genes increased,especially H3K4me3 increased significantly,at the same time maintain low level of repressive histone modifications,leading to high transcriptional activity.While non-lineage specific genes had active histone modifications decreased, but repressive histione modifications level increased,lead to transcription silence.③After CD34~+CD38~- ceils differentiated,the promoter region of HOXA9 had low level of active histone modifications and high level of repressive histone modifications,leading to the gene transcription silencing.更多还原