胚胎干细胞诱导分化过程中组织因子表达及调控的初步研究

发布时间：2018-03-05 16:37

本文选题：人胚胎干细胞　切入点：诱导分化　出处：《中南大学》2012年博士论文　论文类型：学位论文

【摘要】：研究背景近年来基因与蛋白的特异性表达已成为发育生物学中的研究热点。目前已发现多种基因及蛋白活化及其失活在生理发育及疾病发展过程中起着十分重要的调控作用。组织因子(Tissue factor,TF)是由263个氨基酸残基组成的跨膜单链糖蛋白,其在生理及病理情况下的活化及特异性表达对于维持机体凝血平衡和保护重要脏器方面具有不可替代的作用。同时,TF在造血细胞中亦呈现明显的表达差异,其具体调控机制目前尚未明确。表观遗传学作为生物遗传学的重要分支,近年来亦成为生命科学的关注重点。目前认为基因的表观遗传学调控对于基因和蛋白表达以及生物体各类细胞的发育分化起到关键性的作用。同时,人胚胎干细胞(human embryonic stem cells,hESCs)也由于其自身的多向分化潜能和维持自我更新的特性,成为再生医学领域中模拟组织器官发育及探讨生理病理机制的重要实验材料。在这些研究的基础上,我们在本实验中成功建立了胚胎干细胞特异性诱导分化为造血干细胞及滋养层细胞的培养体系,检测了不同分化阶段以及不同类型细胞中TF的表达变化,并进一步探讨了TF在不同细胞种类及各分化阶段的表达是否与其接受表观遗传学调控有关。基于以上目的,本研究分为以下三个章节：第一章hESCs向造血干细胞及滋养层细胞诱导分化体系的建立目的建立一个可行的人胚胎干细胞诱导分化平台,摸索出有效地造血干细胞及滋养层细胞的分化体系,为后续实验提供可靠的细胞标本。方法1.使用胚胎干细胞(hESCs)与小鼠骨髓基质细胞(OP9)共培养方法,诱导分化出HSC细胞,通过RT-PCR及流式细胞学检测细胞特异性标记。2.采用免疫磁珠法分选出CD34+细胞,在体外应用不同细胞因子组合分别诱导CD34+细胞向粒系、红系及巨核系分化,用RT-PCR、流式细胞学检测诱导分化效率。3.使用Marixgel+BMP4方法诱导分化出滋养层细胞,通过RT-PCR及免疫荧光法检测细胞特异性标记。结果1.使用胚胎干细胞(hESCs)与小鼠骨髓基质细胞(OP9)共培养方法,在培养第0天、第5天可见干细胞多能相关基因OCT-4及NANOG表达,造血分化相关基因SCL无表达；培养第9天OCT-4及NANOG无表达,SCL高表达,且流式细胞学检测CD34+细胞比例达到13.89%。2.采用免疫磁珠法分选出CD34+细胞,添加不同细胞因子组合分别诱导14天,流式细胞学检测粒系细胞比例达到93.05%,红系细胞比例达到83.33%,巨核系细胞比例达到68.42%。3.使用Marixgel+BMP4方法诱导分化出滋养层细胞,培养第0天可见OCT-4表达；培养第5天可见滋养层特异性基因CDX2表达,免疫荧光检测示滋养层特异性标记TROMA-1表达。结论我们建立了有效的人胚胎干细胞向造血细胞及滋养层细胞的诱导分化体系,为后续实验提供了前提条件第二章人胚胎干细胞诱导发育过程中组织因子的表达变化目的测定人胚胎干细胞定向发育为造血干细胞、各系血细胞及滋养层细胞过程中组织因子的表达变化。方法1.使用RT-PCR方法检测人胚胎干细胞定向发育过程中TF基因的表达变化。2.使用流式细胞学方法检测TF特异性抗原的表达变化。3.使用TF活性检测TF基因的表达变化。4.使用Western blot检测TF蛋白水平的变化。结果1.在人胚胎干细胞向造血干细胞定向分化过程中,RT-PCR、流式细胞学及TF活性检测均未检测出TF基因表达。2.使用RT-PCR方法,粒系CD15+细胞及巨核系CD41+细胞可检测出TF基因mRNA表达,红系CD235a+细胞未检测出TF基因表达。3.采用流式细胞学检测,粒系细胞、巨核系细胞及红系细胞的TF表达率分别为5.83%、3.94%和1.58%。3.TF活性检测,CD15+细胞可检测到微弱TF活性,其余细胞无明显TF活性表达。4.人胚胎干细胞向滋养层细胞分化过程中可检测出TF的mRNA、蛋白水平及活性表达。结论1.在人胚胎干细胞向造血干细胞发育过程中,无TF及其活性表达。2.在造血干细胞向各系血细胞发育过程中,诱导形成的粒系细胞在mRNA及蛋白水平均表达TF,TF活性微弱表达；巨核系细胞在mRNA水平表达TF基因,但基本无蛋白及活性表达；红系细胞在mRNA及蛋白水平均无TF表达,亦几乎无活性表达。3.在人胚胎干细胞向滋养层细胞分化过程中,诱导形成的滋养层细胞在mRNA及蛋白水平均有TF基因表达,TF活性高表达。第三章hESCs诱导分化过程中TF表观遗传调控的初步研究目的测定人胚胎干细胞定向发育为造血干细胞、各系血细胞及滋养层细胞过程中TF基因DNA启动子区甲基化程度及其变化。方法1.使用特异性甲基化PCR(MSP-PCR)方法检测人胚胎干细胞定向发育过程中TF基因的DNA启动子区甲基化程度及其变化。结果1.在人胚胎干细胞向造血干细胞定向分化过程中,TF基因的DNA启动子区呈现半甲基化半非甲基化表达,并且以甲基化表达为主。2.在造血干细胞定向分化过程中,粒系细胞甲基化程度较前减少,其余各系甲基化及非甲基化程度无明显改变。3.在人胚胎干细胞向滋养层细胞分化过程中,TF基因的DNA启动子去呈现半甲基化半非甲基化表达,并随着滋养层细胞的形成甲基化程度下降,非甲基化程度增加。结论在人胚胎干细胞中TF基因的DNA启动子区呈现半甲基化半非甲基化表达,并随着细胞分化及TF表达增高出现甲基化水平下降,非甲基化水平增加趋势,表明DNA甲基化对于TF基因表达有一定调控作用。
[Abstract]:Research background
In recent years the specific gene and protein expression has become a hot research topic in developmental biology. It has been found that a variety of genes and protein activation and inactivation plays a regulatory role in the development of physiological development and disease is very important. The tissue factor (Tissue factor, TF) is composed of 263 amino acid residues in transmembrane single chain glycoprotein in physiological and pathological conditions and the activation of specific expression plays an irreplaceable role in maintaining balance and protect the important organs of coagulation. At the same time, TF also showed significant differential expression in hematopoietic cells, the specific regulatory mechanism is not yet clear. Epigenetics as an important branch of biological genetics in recent years, has become the focus of life science. Now that the epigenetic regulation of the gene and protein expression in various cells and organisms differentiation gene expression Play a key role. At the same time, human embryonic stem cells (human embryonic stem cells, hESCs) because of its multilineage differentiation potential and maintain self-renewal ability, become the field of regenerative medicine in simulated tissue organ development and pathological mechanisms of physiologically important experimental materials.
On the basis of these studies, we in this experiment successfully established the embryonic stem cell specific differentiation of hematopoietic stem cells and trophoblast cell culture system, detection of changes in different stages of differentiation and the expression of TF in different cell types, and to further explore the expression of TF in different cell types and different stages of differentiation are rather than accept epigenetic regulation. Based on the above purpose, this research is divided into the following three chapters:
The establishment of the first chapter hESCs to hematopoietic stem cells and trophoblast cell differentiation system to establish a viable human embryonic stem cell differentiation platform, explore effective hematopoietic stem cells and trophoblast cell differentiation system, provide a reliable cell samples for subsequent experiments.
1. methods of using embryonic stem cells (hESCs) and bone marrow stromal cells (OP9) co culture method, differentiation of HSC cells, CD34+ cells selected by RT-PCR and flow cytometry cell specific marker.2. by using immunomagnetic beads, in vitro by different cytokine combinations were used to induce CD34+ cells to granulocyte. Erythroid and megakaryocytic differentiation, RT-PCR, flow cytometry was used to detect the differentiation efficiency of.3. Marixgel+BMP4 method is used to induce differentiation of trophoblast cells by detecting cell specific marker RT-PCR and immunofluorescence method.
Results 1. the use of embryonic stem cells (hESCs) and bone marrow stromal cells (OP9) co culture method in culture for zeroth days, fifth days of visible stem cell pluripotency related genes expression in OCT-4 and NANOG, the hematopoietic differentiation related gene SCL expression; cultured for ninth days OCT-4 and NANOG expression, high SCL expression, and flow cytometry cytological detection of CD34+ cells reached 13.89%.2. by immunomagnetic bead sorting CD34+ cells with different cytokine combinations were cultured for 14 days, flow cytometric detection of myeloid ratio reached 93.05%, the proportion of erythroid cells reached 83.33%, the proportion of megakaryocytes reached 68.42%.3. Marixgel+BMP4 method is used to induce differentiation of trophoblast cells cultured for zeroth days of sight the expression of OCT-4 in trophoblast cultured for fifth days; visible specific gene CDX2 expression by immunofluorescence detection of trophoblast specific markers of TROMA-1 expression.
Conclusion we have established an effective induction and differentiation system of human embryonic stem cells to hematopoietic cells and trophoblast cells, which provides the precondition for the follow-up experiment.
Changes in the expression of tissue factors in the second human embryonic stem cell induced development
Objective to determine the changes in the expression of tissue factors in the process of human embryonic stem cell development as hematopoietic stem cells, blood cells and trophoblast cells in various lines.
Changes in expression of.4. expression of.2. TF gene 1. using RT-PCR method for detection of human embryonic stem cells developed in the process of using the expression of.3. by flow cytometry method for detection of TF specific antigen TF gene was detected using TF activity using Western blot to detect the protein level of TF.
Results 1. in human embryonic stem cell differentiation of RT-PCR cells into hematopoietic cells in the process, flow cytometry and TF assay were not detected TF gene expression of.2. using the RT-PCR method, myeloid CD15+ cells and megakaryocytic CD41+ cells can detect the expression of TF gene mRNA, erythroid CD235a+ cells did not detect TF the gene expression of.3. by flow cytometry and myeloid cells, megakaryocytes and erythroid cells, the expression rates of TF were 5.83%, 3.94% and 1.58%.3.TF activity detection, CD15+ cells can detect weak TF activity, the activity of TF cells had no obvious expression of.4. in human embryonic stem cells differentiation into trophoblast cells in detection of TF mRNA, protein expression and activity.
Conclusion 1. in human embryonic stem cells into hematopoietic stem cell development process, TF and activity of.2. expression in hematopoietic stem cells to the development of blood cells in myeloid cells induced expression of TF in both mRNA and protein levels, the activity of TF weak expression; megakaryocyte TF gene expression at the mRNA level no, but the basic activity and protein expression; erythroid cells at mRNA and protein levels showed no TF expression, also almost no active expression of.3. stem cells to differentiation of trophoblast cells in human embryos, the expression induced the formation of trophoblast cells in mRNA and protein levels were high expression of TF gene, the activity of TF.
The preliminary study on epigenetic regulation of TF during the third chapter hESCs induced differentiation
Objective to determine the methylation degree and the change of DNA promoter region of TF gene in hematopoietic stem cells and hematopoietic stem cells.
Methods 1. using specific methylation PCR (MSP-PCR) method to detect the degree of methylation and changes of the TF gene promoter region in the DNA promoter region during the directional development of human embryonic stem cells.
Results 1. in human embryonic stem cells into hematopoietic stem cell differentiation, TF gene DNA promoter methylation in semi semi methylation by methylation and expression, expression of.2. in hematopoietic stem cell differentiation process, myeloid cell methylation level decreased, the rest of the Department of methylation and the non methylation level had no obvious change in.3. stem cells to the differentiation of trophoblast cells in human embryos, the TF gene DNA promoter methylation to present semi semi non methylation expression and methylation level decreased with the formation of trophoblast cells, non methylation level increased.
Conclusion in the human embryonic stem cells, the DNA promoter region of TF gene shows a semi methylated semi and non methylated expression. With the increase of cell differentiation and TF expression, the methylation level decreases, and the level of non methylation increases. This indicates that DNA methylation is related to the expression of TF gene.
A certain regulation effect.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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