本文關(guān)鍵詞：人類多潛能干細(xì)胞體外分化紅細(xì)胞發(fā)育過(guò)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(xì)胞 AGM 紅細(xì)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(xì)胞(human pluripotent stem cells, hPSCs)主要包括人胚胎干細(xì)胞(human embryonic stem cells, hESCs)和人誘導(dǎo)性多潛能干細(xì)胞(human induced pluripotent stem cells, hiPSCs),二者兼具體外自我更新、無(wú)限增殖及多分化潛能的特性。它們的成功建株,極大地推動(dòng)了干細(xì)胞基礎(chǔ)研究和臨床應(yīng)用研究。研究的一個(gè)重要方向是將hPSCs向特定譜系的成熟血液細(xì)胞定向誘導(dǎo)分化。因?yàn)橐恢睕]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(dòng)物模型。hESCs體外誘導(dǎo)分化紅細(xì)胞的過(guò)程,模擬了人類胚胎期體內(nèi)紅系發(fā)生發(fā)育過(guò)程,為研究紅細(xì)胞正常發(fā)育的調(diào)控機(jī)理奠定了實(shí)驗(yàn)基礎(chǔ)。另一方面,利用患者h(yuǎn)iPSCs體系建立體外誘導(dǎo)分化紅細(xì)胞的方法,將為研究紅細(xì)胞早期發(fā)育異常相關(guān)疾病的致病機(jī)理和開發(fā)個(gè)體化治療手段提供理想的平臺(tái)。在所有血細(xì)胞中,成熟紅細(xì)胞因?yàn)椴缓?xì)胞核,并攜帶著最小量的遺傳物質(zhì),壽命較長(zhǎng)等特點(diǎn),有望作為最早的干細(xì)胞來(lái)源的細(xì)胞治療產(chǎn)品而應(yīng)用于臨床。但在成功實(shí)現(xiàn)hPSC來(lái)源的紅細(xì)胞臨床應(yīng)用前,還存在諸多問(wèn)題需解決,例如：培養(yǎng)體系的不健全導(dǎo)致的體外擴(kuò)增效率低、成熟程度低；脫核調(diào)控機(jī)制不明；沒有合適的活體移植模型等。這些困難需要通過(guò)對(duì)hPSC來(lái)源紅細(xì)胞的發(fā)生和成熟過(guò)程中的關(guān)鍵調(diào)控機(jī)理的理解來(lái)攻克。為了精密地研究hPSCs體外誘導(dǎo)分化紅細(xì)胞的發(fā)育過(guò)程,在方法學(xué)上有兩個(gè)亟待解決的問(wèn)題。(1)現(xiàn)有實(shí)驗(yàn)數(shù)據(jù)已顯示不同誘導(dǎo)體系產(chǎn)生的紅細(xì)胞成熟程度有差異。這種差異指向一個(gè)事實(shí),即成體造血微環(huán)境來(lái)源的基質(zhì)細(xì)胞的誘導(dǎo)對(duì)hPSCs產(chǎn)生成熟紅細(xì)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(guò)程產(chǎn)生的成熟紅細(xì)胞。(2) hPSC來(lái)源的紅細(xì)胞分化培養(yǎng)體系中,同時(shí)存在原始造血及成體造血過(guò)程,并且紅細(xì)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(xì)胞,需要建立一種快捷、方便且準(zhǔn)確的標(biāo)識(shí)方法。在成體干細(xì)胞向紅細(xì)胞分化發(fā)育過(guò)程的研究領(lǐng)域,已成功利用表型分子來(lái)區(qū)分紅細(xì)胞的不同發(fā)育階段,啟示我們也可能利用表型分子來(lái)區(qū)分hPSC來(lái)源紅細(xì)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(lái)源的基質(zhì)細(xì)胞,選擇了小鼠主動(dòng)脈-性腺-中腎(Aorta-Gonad-Mesonephros, AGM)細(xì)胞作為共培養(yǎng)體系的基質(zhì)細(xì)胞。AGM區(qū)域是最早的支持成體造血的位點(diǎn)。我們建立了將hPSCs與AGM來(lái)源的細(xì)胞系A(chǔ)GM-S3體外向紅細(xì)胞分化的培養(yǎng)方法。首先將hPSCs細(xì)胞與AGM-S3細(xì)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(xì)胞定向分化并擴(kuò)增。以成體干細(xì)胞hCB-CD34~+來(lái)源的紅細(xì)胞為對(duì)照,用先進(jìn)的多色流式分析技術(shù),探索hPSCs與AGM-S3共培養(yǎng)來(lái)源紅細(xì)胞特異的表型分子表達(dá)譜系。隨后以分別表達(dá)特異表型分子的紅細(xì)胞亞群GPA~+CD36-和GPA~+CD34~+為切入點(diǎn),利用熒光激活細(xì)胞分選(Fluorescence Activated Cell Sorting, FACS)技術(shù),精確地將各目標(biāo)細(xì)胞亞群分選出。然后利用瑞姬氏(May-Grunwald-Giemsa, MGG)染色方法觀察細(xì)胞形態(tài),通過(guò)免疫熒光染色方法考察血紅蛋白組分來(lái)評(píng)價(jià)紅細(xì)胞的成熟程度,并采用qRT-PCR技術(shù)檢測(cè)造血及紅細(xì)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(xì)胞,共培養(yǎng)12天再懸浮培養(yǎng)24天時(shí),紅細(xì)胞數(shù)量約為起始未分化H1細(xì)胞數(shù)量的300倍,其中85%以上表達(dá)成體型血紅蛋白p。hPSC/AGM-S3共培養(yǎng)體系來(lái)源的紅細(xì)胞p血紅蛋白的表達(dá)率遠(yuǎn)遠(yuǎn)高于其他實(shí)驗(yàn)室報(bào)道的數(shù)據(jù)。我們?cè)谶@個(gè)高效體系上進(jìn)一步研究了hPSC/AGM-S3共培養(yǎng)體系來(lái)源紅細(xì)胞發(fā)育過(guò)程的表型分子表達(dá)譜系,發(fā)現(xiàn)共培養(yǎng)階段紅系特有的表型分子GPA陽(yáng)性(GPA~+)細(xì)胞上的其他共表達(dá)表型分子模式與已知的成體型紅細(xì)胞的發(fā)育模式不同。其中GPA和成熟紅細(xì)胞的特定分子CD36和造血干細(xì)胞的特有分子CD34的共表達(dá)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(jìn)行了進(jìn)一步細(xì)致地研究工作。本工作通過(guò)精密細(xì)致地研究,首次發(fā)現(xiàn)并論證了hPSC來(lái)源的早期紅細(xì)胞可根據(jù)GPA和CD36抗原的共表達(dá)的表達(dá)變化指示不同的發(fā)育階段。在共培養(yǎng)階段相當(dāng)長(zhǎng)一段時(shí)間(6-18天)紅細(xì)胞表型為GPA~+CD36-,懸浮培養(yǎng)后逐漸變?yōu)镚PA~+CD36low/~+,10~+5天時(shí)達(dá)到一半左右,然后又再逐漸變?yōu)镚PA~+CD36-。我們?cè)诓煌瑫r(shí)間點(diǎn)將CD36表達(dá)不同的紅細(xì)胞亞群利用流式分選技術(shù)分離出來(lái),通過(guò)對(duì)特定紅細(xì)胞亞群Hb組分、表型分子及基因轉(zhuǎn)錄相對(duì)水平的比較,進(jìn)一步揭示了這一系列的表型變化過(guò)程同時(shí)伴隨著中胚層及內(nèi)皮細(xì)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(xì)胞成熟度逐漸升高等變化過(guò)程。綜合我們的實(shí)驗(yàn)數(shù)據(jù),提示紅細(xì)胞的初期發(fā)育是源于中胚層向內(nèi)皮造血的途徑,遠(yuǎn)在成體造血干／祖細(xì)胞被確定誕生之前。進(jìn)一步的研究還發(fā)現(xiàn)hPSC/AGM-S3共培養(yǎng)階段紅細(xì)胞最初存在于表型為GPA~+CD34~+的細(xì)胞亞群中,細(xì)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(duì)其細(xì)胞形態(tài),人血紅蛋白表達(dá)以及分化潛能進(jìn)行了研究�，F(xiàn)有數(shù)據(jù)顯示GPA~+CD34~+是帶有很強(qiáng)內(nèi)皮細(xì)胞特性的細(xì)胞,約60%表達(dá)人Hb。我們擬進(jìn)一步深入研究GPA~+CD34~+細(xì)胞與內(nèi)皮細(xì)胞的關(guān)系,找出GPA~+CD34~+細(xì)胞的前體細(xì)胞,有望揭示hPSCs細(xì)胞來(lái)源最早紅細(xì)胞的起源和發(fā)育過(guò)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(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R329.2

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6 葉金海;徐袁瑾;顏世果;趙君;涂旗勝;張瑾;段學(xué)靜;Sommer CA;Mostoslavsky G;Kaplan DL;陳錦坤;;SATB2基因轉(zhuǎn)導(dǎo)誘導(dǎo)多潛能干細(xì)胞(iPSCs)修復(fù)小鼠極量顱骨骨缺損[A];2011中國(guó)（威海）干細(xì)胞與組織工程治療前沿論壇論文集[C];2011年

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7 張e，

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