本文選題：人胚胎干細(xì)胞　切入點(diǎn)：誘導(dǎo)分化　出處：《中南大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景 近年來基因與蛋白的特異性表達(dá)已成為發(fā)育生物學(xué)中的研究熱點(diǎn)。目前已發(fā)現(xiàn)多種基因及蛋白活化及其失活在生理發(fā)育及疾病發(fā)展過程中起著十分重要的調(diào)控作用。組織因子(Tissue factor,TF)是由263個氨基酸殘基組成的跨膜單鏈糖蛋白,其在生理及病理情況下的活化及特異性表達(dá)對于維持機(jī)體凝血平衡和保護(hù)重要臟器方面具有不可替代的作用。同時,TF在造血細(xì)胞中亦呈現(xiàn)明顯的表達(dá)差異,其具體調(diào)控機(jī)制目前尚未明確。表觀遺傳學(xué)作為生物遺傳學(xué)的重要分支,近年來亦成為生命科學(xué)的關(guān)注重點(diǎn)。目前認(rèn)為基因的表觀遺傳學(xué)調(diào)控對于基因和蛋白表達(dá)以及生物體各類細(xì)胞的發(fā)育分化起到關(guān)鍵性的作用。同時,人胚胎干細(xì)胞(human embryonic stem cells,hESCs)也由于其自身的多向分化潛能和維持自我更新的特性,成為再生醫(yī)學(xué)領(lǐng)域中模擬組織器官發(fā)育及探討生理病理機(jī)制的重要實(shí)驗(yàn)材料。 在這些研究的基礎(chǔ)上,我們在本實(shí)驗(yàn)中成功建立了胚胎干細(xì)胞特異性誘導(dǎo)分化為造血干細(xì)胞及滋養(yǎng)層細(xì)胞的培養(yǎng)體系,檢測了不同分化階段以及不同類型細(xì)胞中TF的表達(dá)變化,并進(jìn)一步探討了TF在不同細(xì)胞種類及各分化階段的表達(dá)是否與其接受表觀遺傳學(xué)調(diào)控有關(guān)�；谝陨夏康�,本研究分為以下三個章節(jié)： 第一章hESCs向造血干細(xì)胞及滋養(yǎng)層細(xì)胞誘導(dǎo)分化體系的建立目的建立一個可行的人胚胎干細(xì)胞誘導(dǎo)分化平臺,摸索出有效地造血干細(xì)胞及滋養(yǎng)層細(xì)胞的分化體系,為后續(xù)實(shí)驗(yàn)提供可靠的細(xì)胞標(biāo)本。 方法1.使用胚胎干細(xì)胞(hESCs)與小鼠骨髓基質(zhì)細(xì)胞(OP9)共培養(yǎng)方法,誘導(dǎo)分化出HSC細(xì)胞,通過RT-PCR及流式細(xì)胞學(xué)檢測細(xì)胞特異性標(biāo)記。2.采用免疫磁珠法分選出CD34+細(xì)胞,在體外應(yīng)用不同細(xì)胞因子組合分別誘導(dǎo)CD34+細(xì)胞向粒系、紅系及巨核系分化,用RT-PCR、流式細(xì)胞學(xué)檢測誘導(dǎo)分化效率。3.使用Marixgel+BMP4方法誘導(dǎo)分化出滋養(yǎng)層細(xì)胞,通過RT-PCR及免疫熒光法檢測細(xì)胞特異性標(biāo)記。 結(jié)果1.使用胚胎干細(xì)胞(hESCs)與小鼠骨髓基質(zhì)細(xì)胞(OP9)共培養(yǎng)方法,在培養(yǎng)第0天、第5天可見干細(xì)胞多能相關(guān)基因OCT-4及NANOG表達(dá),造血分化相關(guān)基因SCL無表達(dá)；培養(yǎng)第9天OCT-4及NANOG無表達(dá),SCL高表達(dá),且流式細(xì)胞學(xué)檢測CD34+細(xì)胞比例達(dá)到13.89%。2.采用免疫磁珠法分選出CD34+細(xì)胞,添加不同細(xì)胞因子組合分別誘導(dǎo)14天,流式細(xì)胞學(xué)檢測粒系細(xì)胞比例達(dá)到93.05%,紅系細(xì)胞比例達(dá)到83.33%,巨核系細(xì)胞比例達(dá)到68.42%。3.使用Marixgel+BMP4方法誘導(dǎo)分化出滋養(yǎng)層細(xì)胞,培養(yǎng)第0天可見OCT-4表達(dá)；培養(yǎng)第5天可見滋養(yǎng)層特異性基因CDX2表達(dá),免疫熒光檢測示滋養(yǎng)層特異性標(biāo)記TROMA-1表達(dá)。 結(jié)論我們建立了有效的人胚胎干細(xì)胞向造血細(xì)胞及滋養(yǎng)層細(xì)胞的誘導(dǎo)分化體系,為后續(xù)實(shí)驗(yàn)提供了前提條件 第二章人胚胎干細(xì)胞誘導(dǎo)發(fā)育過程中組織因子的表達(dá)變化 目的測定人胚胎干細(xì)胞定向發(fā)育為造血干細(xì)胞、各系血細(xì)胞及滋養(yǎng)層細(xì)胞過程中組織因子的表達(dá)變化。 方法1.使用RT-PCR方法檢測人胚胎干細(xì)胞定向發(fā)育過程中TF基因的表達(dá)變化。2.使用流式細(xì)胞學(xué)方法檢測TF特異性抗原的表達(dá)變化。3.使用TF活性檢測TF基因的表達(dá)變化。4.使用Western blot檢測TF蛋白水平的變化。 結(jié)果1.在人胚胎干細(xì)胞向造血干細(xì)胞定向分化過程中,RT-PCR、流式細(xì)胞學(xué)及TF活性檢測均未檢測出TF基因表達(dá)。2.使用RT-PCR方法,粒系CD15+細(xì)胞及巨核系CD41+細(xì)胞可檢測出TF基因mRNA表達(dá),紅系CD235a+細(xì)胞未檢測出TF基因表達(dá)。3.采用流式細(xì)胞學(xué)檢測,粒系細(xì)胞、巨核系細(xì)胞及紅系細(xì)胞的TF表達(dá)率分別為5.83%、3.94%和1.58%。3.TF活性檢測,CD15+細(xì)胞可檢測到微弱TF活性,其余細(xì)胞無明顯TF活性表達(dá)。4.人胚胎干細(xì)胞向滋養(yǎng)層細(xì)胞分化過程中可檢測出TF的mRNA、蛋白水平及活性表達(dá)。 結(jié)論1.在人胚胎干細(xì)胞向造血干細(xì)胞發(fā)育過程中,無TF及其活性表達(dá)。2.在造血干細(xì)胞向各系血細(xì)胞發(fā)育過程中,誘導(dǎo)形成的粒系細(xì)胞在mRNA及蛋白水平均表達(dá)TF,TF活性微弱表達(dá)；巨核系細(xì)胞在mRNA水平表達(dá)TF基因,但基本無蛋白及活性表達(dá)；紅系細(xì)胞在mRNA及蛋白水平均無TF表達(dá),亦幾乎無活性表達(dá)。3.在人胚胎干細(xì)胞向滋養(yǎng)層細(xì)胞分化過程中,誘導(dǎo)形成的滋養(yǎng)層細(xì)胞在mRNA及蛋白水平均有TF基因表達(dá),TF活性高表達(dá)。 第三章hESCs誘導(dǎo)分化過程中TF表觀遺傳調(diào)控的初步研究 目的測定人胚胎干細(xì)胞定向發(fā)育為造血干細(xì)胞、各系血細(xì)胞及滋養(yǎng)層細(xì)胞過程中TF基因DNA啟動子區(qū)甲基化程度及其變化。 方法1.使用特異性甲基化PCR(MSP-PCR)方法檢測人胚胎干細(xì)胞定向發(fā)育過程中TF基因的DNA啟動子區(qū)甲基化程度及其變化。 結(jié)果1.在人胚胎干細(xì)胞向造血干細(xì)胞定向分化過程中,TF基因的DNA啟動子區(qū)呈現(xiàn)半甲基化半非甲基化表達(dá),并且以甲基化表達(dá)為主。2.在造血干細(xì)胞定向分化過程中,粒系細(xì)胞甲基化程度較前減少,其余各系甲基化及非甲基化程度無明顯改變。3.在人胚胎干細(xì)胞向滋養(yǎng)層細(xì)胞分化過程中,TF基因的DNA啟動子去呈現(xiàn)半甲基化半非甲基化表達(dá),并隨著滋養(yǎng)層細(xì)胞的形成甲基化程度下降,非甲基化程度增加。 結(jié)論在人胚胎干細(xì)胞中TF基因的DNA啟動子區(qū)呈現(xiàn)半甲基化半非甲基化表達(dá),并隨著細(xì)胞分化及TF表達(dá)增高出現(xiàn)甲基化水平下降,非甲基化水平增加趨勢,表明DNA甲基化對于TF基因表達(dá)有 一定調(diào)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.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R329

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