造血干细胞髓系分化中相关基因组蛋白修饰特征的研究

发布时间：2018-06-23 02:35

  本文选题：CD34~+CD38~-细胞 + 细胞分选　； 参考：《中南大学》2009年博士论文

【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。
[Abstract]:The first part is the purification and differentiation of CD34~+CD38~- cells from cord blood in vitro.
Objective: to establish a feasible method for the selection of CD34~+CD38~- cells from umbilical cord blood, and to find out the effective granulocyte, red and megakaryocyte differentiation system in vitro, and provide reliable cell specimens for subsequent experiments.
Methods: (1) CD34~+ cells were selected by magnetic activated cell sorting (MACS), CD34~+CD38~- cells were selected by two negative sorting, and the purity of the cells were detected by flow cytometry and the cell viability was detected by trypan blue staining. (2) the group of SCF+IL-3+G-CSF or EPO or TPO cytokines was used in vitro. The cells of CD34~+CD38~- were induced to differentiate into granulocyte, red and megakaryocytes respectively. The cell proliferation curves were plotted by cell counting and the efficiency of differentiation by flow cytometry was used to detect the differentiation.
Results: after the first separation of CD34~+ cells with anti CD34 magnetic beads, the purity of CD34~+ cells could reach 95.24 + 1.03%. After the second separation, the cell viability of the CD34~+/CD38~- cells was more than 99% before and after the separation of 90.23 + 2.52%.. (2) the number of fine cell lines increased by 1186.67 + 106.1 times, and the number of erythroid cells increased. The number of megakaryocyte cells increased by 627 + 49.65 times by 894.67 + 48.22 times. The results of flow cytometry showed that the ratio of CD15~+ cells was 91.49%, the ratio of CD235a~+ cells was 95.55% and the ratio of CD41a~+ cells was 86.52%. in the fourteenth days of induced differentiation.
Conclusion: we have established an effective MACS sorting method and an in vitro induction method, which provides reliable source of cell specimens for our subsequent experiments.
The second part is the establishment of micro chromatin immunoprecipitation method.
Objective: chromatin immunoprecipitation assay (ChIP) is one of the powerful techniques to study the interaction of protein and DNA. However, the maximum number of cells in the current ChIP test method requires a large number of cells, and the cells of our separation are difficult to reach this requirement. Therefore, our aim is to establish a kind of energy. The ChIP method in a few cells is called small chromatin immunoprecipitation (miniChIP).
Methods: the miniChIP experimental method was established on the basis of the traditional ChIP method, and the level of histone 4 (acH4) in the different loci of the beta globin gene expressed in MEL cells before and after induction was studied by using the traditional ChIP experiment method and the miniChIP experiment on the basis of the traditional method. The reliability and specificity of the new miniChIP method, including the Gao Min locus 2 (HS2), the promoter region of the beta maj gene and the promoter region of the Ey gene, are found in the new method.
Results: in the untreated MEL cells, a certain acH4 level existed in the promoter region of the HS2 and beta maj genes with the miniChIP method, while the Ey gene promoter region detected that the acH4 level.MEL cells of the extremely low level of.MEL were induced, and the acH4 level of the HS2 and beta maj gene promoter regions was greatly increased by 2.8, respectively. The acH4 level in the promoter region of the 7 and 2.26 times.Ey gene was almost unchanged. This was in agreement with the experimental results observed by the traditional ChIP experiment and the results obtained by other researchers in the traditional ChIP experiment.
Conclusion: Based on the traditional ChIP experimental method, a ChIP test method, called miniChIP., which can be used in a small number of cells, was established and verified in the MEL cells before and after induction. The feasibility and reliability of the miniChIP method were confirmed.
The third part is about the histone modification characteristics of hematopoietic stem cells during myeloid differentiation.
Objective: To observe the histone modification characteristics of the transcription factors and genes related to hematopoietic differentiation in CD34~+CD38~- cells and in the differentiated cells, and to explore the possible role of chromatin conformation in the maintenance and differentiation of hematopoietic stem cells in multipotential and lineage differentiation.
Methods: qRT-PCR was used to detect the mRNA expression level of hematopoietic differentiation related transcription factors and genes in different types of cells. Different types of cells include CD34~+CD38~- cells and induced CD15~+ cells, CD235a~+ cells, CD41a~+ cells. Hematopoietic differentiation related transcription factors and genes include early hematopoietic transcription related transcription. Factor HOXA9, granulocyte differentiation related transcription factor PU.1, granulocyte specific gene MPO, CD11b, erythroid megakaryocyte differentiation related transcription factor GATA-1, erythroid specific gene EPOR and megakaryocytic specific gene CD41a: lymphoid differentiation related transcription factor GATA-3, PAX5, lymphoid specific gene CD3, CD79a. 2, we use miniChIP-qPCR experimental methods in different types. The changes in the 6 histone modifications of the promoter region of the hematopoietic differentiation related transcription factors and genes were observed and compared. The 6 different histone modifications were activated histone modification including histone 3 acetylation (acH3), histone 4 acetylation (acH4), histone 3 fourth lysine two methylation (H3K4me2), histone 3 The trimethylation (H3K4me3) and the inhibitory histone modification of fourth lysine include trimethylation (H3K9me3) of histone 3 ninth bits lysine, and histone 3 twenty-seventh methylation of lysine (H3K27me3).
Results: (1) there was a low level of mRNA expression or non expression in the differentiation related transcription factors and genes in the CD34~+CD38~- cells. The expression of the transcription factor HOXA9 related to the early hematopoiesis was highly expressed. When the CD34~+CD38~- cells differentiated into the granulocyte specific differentiation, the expression of PU.1, MPO, CD11b in the granulocyte related genes increased obviously; CD34~+CD38~- cells turned to the red system. After specific differentiation, the expression of GATA-1 and EPOR increased significantly, and the expression of megakaryocyte related genes GATA-1, CD41a expression was significantly increased after the specific differentiation of CD34~+CD38~- cells to megakaryocyte, and the non lineage related genes were not detected and the expression of HOXA9 gene decreased significantly after the specific differentiation of CD34~+CD38~- cell lines; and (2) in CD34~+CD38~- cells The transcriptional factors and genes of differentiation in each line have a certain level of acH4 modification and slightly low level of acH3 modification and high level of H3K4me2 modification, but the level of H3K4me3 modification is very low. (3) the transcription factors and genes of each lineage differentiation in CD34~+CD38~- cells are modified with low level H3K9me3 and H3K27me3; (4) along with CD34~+C After the specific differentiation of D38~- cell lines, the level of acH3, acH4 and H3K4me2 of the specific genes increased slightly, but the level of H3K4me3 increased significantly, while H3K9me3 and H3K27me3 modification remained at a low level. The level of acH3 and acH4 modification of non specific genes decreased, and H3K4me3 modification remained at a low level, while H3K9me3 or / and H3K27me3 levels were at the same time. After the differentiation of CD34~+CD38~- cells to terminal cells, the activation histone modification on the promoter region of the early hematopoiesis related transcription factor HOXA9, including acH3, acH4, H3K4me2, H3K4me3, was significantly reduced, while the inhibitory histone modification including H3K9me3 and H3K27me3 increased significantly. Conclusion: (1) CD34+C rich in hematopoietic stem cells The genes related to differentiation of D38- cells have a certain level of H3, acetylation of H4, high level of H3K4me2 modification, low level H3K4me3 modification and low level histone inhibition. These genes are expressed at low level or not detected. 2. After the specific differentiation of CD34+ CD38- cell lines, the a of the related genes The level of cH3, acH4, H3K4me2 modification remained unchanged or slightly increased, but the level of H3K4me3 modification increased significantly, while the low level of inhibitory histone modification was maintained, and the gene expression was high transcriptional, while the acH3, acH4 modification level of the non line related genes was lower and the H3K4me3 level remained at a low level, but the enrichment of the H3K4me3 level was at a low level. After the differentiation of CD34+CD38- cells to terminal cells, the level of H3K4me3 modification on the promoter region of the early hematopoietic gene HOXA9 decreased, but the inhibitory histone modification markers increased significantly, showing the silence of gene expression.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329

【共引文献】

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