本文關(guān)鍵詞：人類多潛能干細胞體外分化紅細胞發(fā)育過程中表型分子的研究　出處：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人類多潛能干細胞 AGM 紅細胞發(fā)育 原始/成體造血 CD36 CD34

【摘要】：研究目的：人類多潛能干細胞(human pluripotent stem cells, hPSCs)主要包括人胚胎干細胞(human embryonic stem cells, hESCs)和人誘導(dǎo)性多潛能干細胞(human induced pluripotent stem cells, hiPSCs),二者兼具體外自我更新、無限增殖及多分化潛能的特性。它們的成功建株,極大地推動了干細胞基礎(chǔ)研究和臨床應(yīng)用研究。研究的一個重要方向是將hPSCs向特定譜系的成熟血液細胞定向誘導(dǎo)分化。因為一直沒有合適的研究人類早期造血發(fā)生的模型,以往的研究主要都是基于小鼠等動物模型。hESCs體外誘導(dǎo)分化紅細胞的過程,模擬了人類胚胎期體內(nèi)紅系發(fā)生發(fā)育過程,為研究紅細胞正常發(fā)育的調(diào)控機理奠定了實驗基礎(chǔ)。另一方面,利用患者hiPSCs體系建立體外誘導(dǎo)分化紅細胞的方法,將為研究紅細胞早期發(fā)育異常相關(guān)疾病的致病機理和開發(fā)個體化治療手段提供理想的平臺。在所有血細胞中,成熟紅細胞因為不含細胞核,并攜帶著最小量的遺傳物質(zhì),壽命較長等特點,有望作為最早的干細胞來源的細胞治療產(chǎn)品而應(yīng)用于臨床。但在成功實現(xiàn)hPSC來源的紅細胞臨床應(yīng)用前,還存在諸多問題需解決,例如：培養(yǎng)體系的不健全導(dǎo)致的體外擴增效率低、成熟程度低；脫核調(diào)控機制不明；沒有合適的活體移植模型等。這些困難需要通過對hPSC來源紅細胞的發(fā)生和成熟過程中的關(guān)鍵調(diào)控機理的理解來攻克。為了精密地研究hPSCs體外誘導(dǎo)分化紅細胞的發(fā)育過程,在方法學(xué)上有兩個亟待解決的問題。(1)現(xiàn)有實驗數(shù)據(jù)已顯示不同誘導(dǎo)體系產(chǎn)生的紅細胞成熟程度有差異。這種差異指向一個事實,即成體造血微環(huán)境來源的基質(zhì)細胞的誘導(dǎo)對hPSCs產(chǎn)生成熟紅細胞是必需的。所以需要建立一套高效并趨于自然的共培養(yǎng)方法,以得到類似于自然發(fā)育過程產(chǎn)生的成熟紅細胞。(2) hPSC來源的紅細胞分化培養(yǎng)體系中,同時存在原始造血及成體造血過程,并且紅細胞在早期發(fā)育存在不同的發(fā)育階段。為了辨別不同的細胞,需要建立一種快捷、方便且準(zhǔn)確的標(biāo)識方法。在成體干細胞向紅細胞分化發(fā)育過程的研究領(lǐng)域,已成功利用表型分子來區(qū)分紅細胞的不同發(fā)育階段,啟示我們也可能利用表型分子來區(qū)分hPSC來源紅細胞的早期不同發(fā)育階段。研究方法：我們比較了不同的成體造血微環(huán)境來源的基質(zhì)細胞,選擇了小鼠主動脈-性腺-中腎(Aorta-Gonad-Mesonephros, AGM)細胞作為共培養(yǎng)體系的基質(zhì)細胞。AGM區(qū)域是最早的支持成體造血的位點。我們建立了將hPSCs與AGM來源的細胞系A(chǔ)GM-S3體外向紅細胞分化的培養(yǎng)方法。首先將hPSCs細胞與AGM-S3細胞系先共培養(yǎng)誘導(dǎo)造血分化的發(fā)生,再經(jīng)懸浮培養(yǎng)向紅細胞定向分化并擴增。以成體干細胞hCB-CD34~+來源的紅細胞為對照,用先進的多色流式分析技術(shù),探索hPSCs與AGM-S3共培養(yǎng)來源紅細胞特異的表型分子表達譜系。隨后以分別表達特異表型分子的紅細胞亞群GPA~+CD36-和GPA~+CD34~+為切入點,利用熒光激活細胞分選(Fluorescence Activated Cell Sorting, FACS)技術(shù),精確地將各目標(biāo)細胞亞群分選出。然后利用瑞姬氏(May-Grunwald-Giemsa, MGG)染色方法觀察細胞形態(tài),通過免疫熒光染色方法考察血紅蛋白組分來評價紅細胞的成熟程度,并采用qRT-PCR技術(shù)檢測造血及紅細胞發(fā)育相關(guān)的重要基因的轉(zhuǎn)錄水平。研究結(jié)果：我們建立了高效的hPSC/AGM-S3共培養(yǎng)造血誘導(dǎo)分化體系,可以產(chǎn)生大量的高純度和高成熟度紅細胞,共培養(yǎng)12天再懸浮培養(yǎng)24天時,紅細胞數(shù)量約為起始未分化H1細胞數(shù)量的300倍,其中85%以上表達成體型血紅蛋白p。hPSC/AGM-S3共培養(yǎng)體系來源的紅細胞p血紅蛋白的表達率遠遠高于其他實驗室報道的數(shù)據(jù)。我們在這個高效體系上進一步研究了hPSC/AGM-S3共培養(yǎng)體系來源紅細胞發(fā)育過程的表型分子表達譜系,發(fā)現(xiàn)共培養(yǎng)階段紅系特有的表型分子GPA陽性(GPA~+)細胞上的其他共表達表型分子模式與已知的成體型紅細胞的發(fā)育模式不同。其中GPA和成熟紅細胞的特定分子CD36和造血干細胞的特有分子CD34的共表達均存在hPSCs分化發(fā)育的特有模式。我們根據(jù)這兩條線索,進行了進一步細致地研究工作。本工作通過精密細致地研究,首次發(fā)現(xiàn)并論證了hPSC來源的早期紅細胞可根據(jù)GPA和CD36抗原的共表達的表達變化指示不同的發(fā)育階段。在共培養(yǎng)階段相當(dāng)長一段時間(6-18天)紅細胞表型為GPA~+CD36-,懸浮培養(yǎng)后逐漸變?yōu)镚PA~+CD36low/~+,10~+5天時達到一半左右,然后又再逐漸變?yōu)镚PA~+CD36-。我們在不同時間點將CD36表達不同的紅細胞亞群利用流式分選技術(shù)分離出來,通過對特定紅細胞亞群Hb組分、表型分子及基因轉(zhuǎn)錄相對水平的比較,進一步揭示了這一系列的表型變化過程同時伴隨著中胚層及內(nèi)皮細胞特性的丟失,原始造血向成體造血特性的轉(zhuǎn)變以及紅細胞成熟度逐漸升高等變化過程。綜合我們的實驗數(shù)據(jù),提示紅細胞的初期發(fā)育是源于中胚層向內(nèi)皮造血的途徑,遠在成體造血干／祖細胞被確定誕生之前。進一步的研究還發(fā)現(xiàn)hPSC/AGM-S3共培養(yǎng)階段紅細胞最初存在于表型為GPA~+CD34~+的細胞亞群中,細胞亞群比例先增加后減少。流式分選出共培養(yǎng)10天中的GPA~+CD34~+,對其細胞形態(tài),人血紅蛋白表達以及分化潛能進行了研究�，F(xiàn)有數(shù)據(jù)顯示GPA~+CD34~+是帶有很強內(nèi)皮細胞特性的細胞,約60%表達人Hb。我們擬進一步深入研究GPA~+CD34~+細胞與內(nèi)皮細胞的關(guān)系,找出GPA~+CD34~+細胞的前體細胞,有望揭示hPSCs細胞來源最早紅細胞的起源和發(fā)育過程。
[Abstract]:Objective: To study human pluripotent stem cells (human pluripotent stem cells, hPSCs) mainly includes human embryonic stem cells (human embryonic stem cells, hESCs) and human induced pluripotent stem cells (human induced pluripotent stem cells, hiPSCs), two with self-renewal, unlimited proliferation characteristics and multi differentiation potential. Their success were greatly promoted the stem cell research and clinical application research. An important research direction is to induce hPSCs differentiate to specific lineages of mature blood cells. Because there has been no research orientation of early human hematopoiesis model is appropriate, the previous studies are mainly the process of differentiation of red blood cells animal model of.HESCs in vitro based on Simulation of the human embryo in vivo erythroid development process, lay the regulation mechanism for the normal development of red blood cells Based. On the other hand, establishment method of differentiation of red blood cells in vitro by patients with hiPSCs system, pathogenic mechanism and development of individualized treatment means abnormal development of related diseases for early studies of red blood cells provide an ideal platform. In all blood cells, mature red blood cells because of not containing the nucleus, and carrying the genetic material minimum the long life and other characteristics, is expected as a cell therapy product of the earliest source of stem cells for clinical application. But in clinical application of red blood cells in the successful implementation of hPSC source, there are still many problems need to be solved, such as: the training system is not perfect in the in vitro amplification of low efficiency, low degree of maturity; deribonucleic the molecular mechanism is still unknown; there is no suitable living donor transplantation model. These difficulties through the key regulatory mechanism and process of mature red blood cells in the hPSC source of understanding to overcome. The development process of differentiation of red blood cells on the precision of hPSCs in vitro, the method has two problems to be solved on the school. (1) the existing experimental data have shown that there is a difference in degrees of maturity have different induction system of red blood cells. These differences point to the fact that induce stromal cells in hematopoietic microenvironment of the source body the mature red blood cells on hPSCs is required. So it is necessary to establish a set of effective and tends to natural co culture method to obtain similar mature red blood cells in the natural development process produced. (2) red cell differentiation from hPSC culture system, at the same time are primitive hematopoietic and adult hematopoiesis, and red cells in the early development of different developmental stages. In order to identify the different types of cells, we need to establish a fast, convenient and accurate identification method. In the study of adult stem cells in the red cell differentiation process. Domain, different developmental stages have been successfully used to cell phenotypic molecules into different developmental stages, early enlightenment we may also use phenotypic molecules to differentiate hPSC from red blood cells. Methods: We compared the stromal cell body of hematopoietic microenvironment into different sources, the mouse aorta gonad mesonephros (Aorta-Gonad-Mesonephros, AGM) cells as co cultured stromal cells.AGM system is the first to support hematopoietic sites. We established the culture square hPSCs and AGM derived cell lines AGM-S3 in vitro erythroid differentiation method. Firstly, hPSCs cells and AGM-S3 cells to co culture induction of hematopoietic differentiation occurred, followed by suspension culture of red cell differentiation and amplification. In adult stem cells from the red blood cells of hCB-CD34~+ were type analysis technology with advanced multi-color flow, exploring hPSCs and AGM-S3 Cultured erythroid specific sources of phenotypic molecules. Then the expression of lineage specific expression to phenotypic molecules of red blood cell subsets of GPA~+CD36- and GPA~+CD34~+ as the starting point, using fluorescence activated cell sorting (Fluorescence Activated Cell Sorting FACS) technology, precisely the target cell subsets were selected. Then the other (May-Grunwald-Giemsa, MGG) the cell morphology was observed by staining method, investigation group to evaluate the degree of maturity of hemoglobin in red blood cells by immunofluorescence staining, and the transcription level of qRT-PCR detection of hematopoietic and red blood cell development important genes. Results: we established a highly efficient hPSC/AGM-S3 hematopoietic differentiation of co culture system, can produce a large amount of high purity and the high maturity of red blood cells, co cultured 12 days suspension cultured for 24 days, the number of red blood cells about initiation of undifferentiated H1 cells 300 times the number of more than 85% of them expressed as the expression system of source of co culture of hemoglobin p.hPSC/AGM-S3 red blood cell p type hemoglobin rate is much higher than other reported laboratory data. In this system, we studied the hPSC/AGM-S3 co culture during the development of the source of red cell phenotypic molecules expression system of lineage, found co cultured erythroid specific the molecular phenotype of GPA positive cells (GPA~+) on the other co expression of phenotypic molecules known as development mode and mode shape of red blood cells. Different unique patterns of differentiation and development of hPSCs were co expressed in the specific molecular GPA and mature red blood cells CD36 and hematopoietic stem cell specific molecules CD34. According to the two clues for further detailed research work. In this work, through meticulous precision, first discovered and demonstrated early hPSC sources Red blood cells may indicate different developmental stages according to the changes of the expression of the co expression of GPA and CD36 antigen. In the co culture stage for quite a long period of time (6-18 days) red blood cell phenotype was GPA~+CD36- after suspension culture has gradually changed to GPA~+CD36low/~+, 10~+5 days to about half, and then gradually changed to GPA~+CD36-. we are in different the expression of CD36 in different time points of red blood cell subsets by flow cytometry analysis separated by specific red cell subsets in Hb group, compared the molecular phenotypes and gene transcription relative level, further reveals the phenotypic changes in the process of this series is accompanied by loss of mesoderm and endothelial cells into primitive hematopoietic characteristics. Change characteristics of hematopoietic and red cell maturity gradually rise higher. Comprehensive change process of our experimental data, suggesting that early red blood cell development is derived from mesoderm inward leather Blood way before adult hematopoietic stem / progenitor cells was determined. The birth of further research found that hPSC/AGM-S3 co culture stage of red blood cells initially present in the phenotype of GPA~+CD34~+ cell subsets, the proportion of cell subsets increased firstly and then decreased. Flow cytometry sorting out co cultured for 10 days in GPA~+CD34~+, on the cell morphology, expression of human hemoglobin and differentiation potential were studied. The existing data show that GPA~+CD34~+ is a strong endothelial cells, about 60% Hb. expression we intend to further study of GPA~+CD34~+ cells and endothelial cells, progenitor cells found in GPA~+CD34~+ cells, the process is expected to reveal hPSCs cells derived from the earliest origin of red blood cells and development.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R329.2

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6 李磊;人胚胎多潛能干細胞向胰島素分泌細胞轉(zhuǎn)化的研究[D];吉林大學(xué);2009年

7 張e，

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