本文選題：人類多潛能干細(xì)胞(hPSCs) + AGM-S3��； 參考：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文

【摘要】：研究背景和目的:肥大細(xì)胞(Mast cells,MC)和巨噬細(xì)胞(Macrophage,MΦ)是非常重要的免疫細(xì)胞,在機(jī)體固有免疫反應(yīng)中具有舉足輕重的作用。MC和MΦ具有極大的異質(zhì)性。在人類MΦ既有分布于血液中以單核形式存在的狀態(tài),也有在多種組織和器官中分布的組織駐留型,并根據(jù)分布的形態(tài)和功能具有不同命名;而人類MC根據(jù)顆粒中含有蛋白酶種類不同,分為MCT和MCTc兩類,前者與小鼠黏膜MC相似,后者與小鼠組織相關(guān)MC類似。MΦ和MC表面和內(nèi)體分布多種Toll樣受體(TLRs,一種機(jī)體內(nèi)重要的模式識(shí)別受體),可有效識(shí)別病原體相關(guān)外源性危險(xiǎn)信號(hào)(PAMPs)和自身細(xì)胞所釋放的內(nèi)源性危險(xiǎn)信號(hào)(DAMPs),在機(jī)體防御中發(fā)揮著重要的作用。在個(gè)體發(fā)生的早期階段,血細(xì)胞起源在順序上有著時(shí)空分布的不同特征。所有血細(xì)胞均來(lái)源于中胚層,最早的造血細(xì)胞起源于卵黃囊;隨后進(jìn)入胚內(nèi)的主動(dòng)脈-性腺-中腎區(qū)域(AGM區(qū)),在此可產(chǎn)生最初的能夠確認(rèn)的造血干細(xì)胞;最后進(jìn)入胎肝造血,在此含有大量可移植的造血干細(xì)胞。依據(jù)人體造血發(fā)生的時(shí)空順序及不同特征,可將其分為原始造血和成體造血。在成體造血,所有功能型血細(xì)胞均來(lái)源于骨髓中的造血干細(xì)胞(Hematopoietic stem cells,HSCs)。然而,對(duì)小鼠胚胎造血發(fā)育的研究顯示,起源于卵黃囊的原始造血以及紅系/髓系祖細(xì)胞(EMP)都可產(chǎn)生MΦ。另外,在小鼠胎肝發(fā)育過(guò)程中發(fā)現(xiàn)MC前體高度集中于卵黃囊和胎肝血中,提示MΦ和MC存在一個(gè)較強(qiáng)的早期胚性發(fā)育階段。研究發(fā)現(xiàn),產(chǎn)生于早期胚性發(fā)育階段的這兩種固有免疫細(xì)胞具有組織偏向特性。由于受倫理道德及法律的限制,在人體中進(jìn)行胚胎造血發(fā)育的研究存在諸多困難,導(dǎo)致對(duì)人類早期胚胎造血發(fā)生認(rèn)識(shí)的缺失。對(duì)造血細(xì)胞發(fā)生學(xué)的研究主要基于小鼠等動(dòng)物模型。但是動(dòng)物體內(nèi)的研究數(shù)據(jù)并不能完全指示人體內(nèi)的發(fā)育過(guò)程。隨著人胚胎干細(xì)胞(human embryonic stem cells,hESCs)的成功建系以及人誘導(dǎo)性多能干細(xì)胞(human induced pluripotent stem cells,hiPSCs)的體外重編程獲得,為研究人類早期發(fā)育機(jī)制提供了有效的手段。hESCs和hiPSCs統(tǒng)稱為人多能干細(xì)胞(human pluripotent stem cells,hPSCs),具有自我更新、無(wú)限增殖及分化形成完整個(gè)體的能力,為研究跨胚層細(xì)胞、組織以及所有成體細(xì)胞的發(fā)育,提供了有效的手段。已有大量關(guān)于從hPSCs體外成功分化出三個(gè)胚層來(lái)源成熟細(xì)胞的報(bào)道�；趆PSCs向造血細(xì)胞誘導(dǎo)分化的研究,可很大程度上重現(xiàn)早期人類造血發(fā)生過(guò)程,有助于詳盡地解析人類胚胎期造血的發(fā)育模式。為了研究人類早期發(fā)育過(guò)程中MC和MΦ的發(fā)育情況,從而更好地模擬體內(nèi)早期發(fā)育,需要在體外建立一種高效地趨于自然發(fā)育過(guò)程的培養(yǎng)體系。通過(guò)與成體HSCs來(lái)源固有免疫細(xì)胞比較,我們研究了早期造血發(fā)育產(chǎn)生固有免疫細(xì)胞的功能及識(shí)別能力。在此,我們以人固有免疫細(xì)胞初期發(fā)育作為研究方向,重點(diǎn)探索hPSCs體外誘導(dǎo)產(chǎn)生MC和MΦ初期發(fā)育的起源及其功能。研究方法:基于hPSCs/AGM-S3共培養(yǎng)體系,建立固有免疫細(xì)胞體外培養(yǎng)方法。以MC和MΦ為主,分別檢測(cè)兩種固有免疫細(xì)胞的形態(tài)、表型特征以及功能,并探索兩種細(xì)胞的起源。具體操作方法如下:1.以小鼠主動(dòng)脈-性腺-中腎(Aorta-Gonad-Mesonephros,AGM)來(lái)源基質(zhì)細(xì)胞系(AGM-S3),與hPSCs體外共培養(yǎng),誘導(dǎo)產(chǎn)生大量造血干/祖細(xì)胞,進(jìn)一步懸浮培養(yǎng)定向產(chǎn)生MΦ和MC。培養(yǎng)方法分為三步:1)將hPSCs細(xì)胞接種到AGM-S3上進(jìn)行共培養(yǎng),誘導(dǎo)造血分化發(fā)生,監(jiān)測(cè)不同時(shí)間點(diǎn)克隆形態(tài)及表型分子表達(dá)變化,2)經(jīng)懸浮培養(yǎng)一周,擴(kuò)增造血干/祖細(xì)胞,3)更換培養(yǎng)基進(jìn)行MC或MΦ定向分化。2.觀察兩種固有免疫細(xì)胞形態(tài)及表型特征1)MC形態(tài)學(xué)特征:麥格氏-吉姆薩(May-GrunwaldGiemsa,MGG)、酸性甲苯胺藍(lán)(Acidictoluidineblue)以及阿爾新藍(lán)(Alcian blue)化學(xué)染色觀察細(xì)胞形態(tài);電鏡觀察MC內(nèi)顆粒形態(tài);免疫熒光染色觀察MC中Carboxypeptidase A、Cathepsin-G、Tryptase和Chymase蛋白酶的表達(dá)情況,多色流式檢測(cè)技術(shù)分析MC表型分子表達(dá)譜系。2)MΦ形態(tài)學(xué)特征:MGG染色觀察三種來(lái)源MΦ形態(tài);多色流式檢測(cè)技術(shù)分析三種來(lái)源MΦ表型分子表達(dá)譜系。3.檢測(cè)兩種固有免疫細(xì)胞功能1)MC功能檢測(cè):酶聯(lián)免疫吸附法檢測(cè)IgE、Substance P和Compound48/80刺激下,MC上清及細(xì)胞沉淀中組胺的含量;RT-qPCR方法檢測(cè)TLRs表達(dá)。2)Φ功能檢測(cè):利用RT-qPCR方法檢測(cè)不同極化條件下,炎癥因子及TLRs表達(dá);通過(guò)低密度脂蛋白(LDL)吞噬實(shí)驗(yàn)檢測(cè)MΦ吞噬能力。4.尋找MC的祖細(xì)胞:利用流式分選技術(shù)(Fluorescence Activated Cell Sorting,FACS)分選目標(biāo)細(xì)胞群,確定MC祖細(xì)胞結(jié)果:1.建立高效的hPSCs/AGM-S3共培養(yǎng)造血細(xì)胞誘導(dǎo)分化體系,可產(chǎn)生大量高純度MC和初期MΦ。1)共培養(yǎng)14天再向MC定向培養(yǎng)10天,體系中發(fā)現(xiàn)C-KIT+,CD45+,Tryptase+和Chymase+的MC,40天雙陽(yáng)比例高達(dá)98%。2)共培養(yǎng)3天再向MΦ定向培養(yǎng)14天,可產(chǎn)生初期MΦ。2.產(chǎn)生的兩種固有免疫細(xì)胞具有組織偏向型1)化學(xué)染色,免疫熒光染色,電鏡觀察以及IgE、SubstanceP和Compound48/80刺激下,MC都可釋放組胺,確定我們的培養(yǎng)體系產(chǎn)生的MC為MCTC類型。2)初期產(chǎn)生MΦ在IL-4刺激可有效極化成M2型3.產(chǎn)生的兩種固有免疫細(xì)胞與成體HSCs來(lái)源固有免疫細(xì)胞不同1)以外周血單個(gè)核細(xì)胞作為對(duì)照,與臍帶血CD34+細(xì)胞來(lái)源的MC相比,hESCs/AGM-S3共培養(yǎng)產(chǎn)生的MC高表達(dá)TLR2,TLR4和TLR9。2)與臍帶血CD34+細(xì)胞來(lái)源MΦ以及共培養(yǎng)14天來(lái)源MΦ相比,共培養(yǎng)3天來(lái)源MΦ表型分子具有明顯不同的表達(dá)水平。IL-4刺激共培養(yǎng)3天來(lái)源MΦ中TLR1～TLR8基因表達(dá)水平明顯高于前兩種來(lái)源MΦ。4.產(chǎn)生的兩種固有免疫細(xì)胞誕生于成體造血干/祖細(xì)胞(CD34+CD45+)之前1)流式分選共培養(yǎng)第8天CD34+C-KIT-和CD34+C-KIT+兩群細(xì)胞,向MC定向培養(yǎng)14天,免疫熒光染色顯示兩亞群產(chǎn)生子代細(xì)胞Tryptase和Chymase雙陽(yáng)百分比都大于85%。2)共培養(yǎng)第3天產(chǎn)生的MΦD,誕生于成體造血干/祖細(xì)胞之前。結(jié)論:1.hPSCs/AGM-S3造血共培養(yǎng)體系可誘導(dǎo)產(chǎn)生MC和MΦ等固有免疫細(xì)胞,并且在成體造血干/祖細(xì)胞(CD34+CD45+細(xì)胞)誕生之前,可產(chǎn)生一群MC和MΦ。2.這群MC和MΦ具有自己獨(dú)特的發(fā)育途徑。3.這些初期產(chǎn)生的固有免疫細(xì)胞偏向于組織特性,高表達(dá)TLRs,說(shuō)明分布于組織中的MC和MΦ具有更強(qiáng)的免疫識(shí)別能力,在固有免疫反應(yīng)中發(fā)揮著重要的作用。
[Abstract]:Background and purpose: Mast cells (MC) and macrophages (Macrophage, M diameter) are very important immune cells, which play an important role in the inherent immune response of the body..MC and M are of great heterogeneity. In human M, the human M is distributed in the form of mononuclear in the blood, and in a variety of tissues and organs. The tissue residency type in the middle distribution is named according to the morphology and function of the distribution, and human MC is divided into two classes of MCT and MCTc according to the variety of protease in the particles. The former is similar to the mouse mucous membrane MC. The latter is similar to the mouse tissue, and the MC is similar to the.M diameter and the MC surface and the inner body distribution of various Toll like receptors (TLRs, an important body within the body. The pattern recognition receptor) can effectively identify the exogenous risk signals (PAMPs) and endogenous risk signals (DAMPs) released by their own cells and play an important role in the body's defense. In the early stages of the individual occurrence, the origin of blood cells has different characteristics in the sequence of spatiotemporal distribution. All blood cells are derived from all blood cells. Mesoderm, the earliest hematopoietic cells originate from the yolk sac; then enter the aorta of the embryo - the gonadal - mesarenal region (AGM region), which can produce the original confirmed hematopoietic stem cells; finally, it enters the fetal liver and hematopoiesis, which contains a large number of transplanted hematopoietic stem cells. It is divided into primary hematopoiesis and adult hematopoiesis. In adult hematopoiesis, all functional hemocytes are derived from Hematopoietic stem cells (HSCs) in the bone marrow. However, the study of hematopoietic development in mouse embryos shows that the original hematopoiesis originating from the yolk sac and the erythroid / myeloid progenitor cells (EMP) can produce M diameter. In addition, in the mouse fetal liver During the development, the MC precursor was found to be highly concentrated in the yolk sac and fetal liver blood, suggesting that there is a strong early embryonic development stage in M diameter and MC. The study found that the two innate immune cells produced in the early embryonic development stage have the characteristics of tissue bias. There are many difficulties in the study of blood development, which leads to the lack of understanding of human early embryo hematopoiesis. The study of hematopoiesis is mainly based on mice and other animal models. However, the research data in animals do not fully indicate the development process in the human body. With human embryonic stem cells (Human embryonic stem cells, hESCs) In vitro reprogramming of successful construction and human induced pluripotent stem cells (human induced pluripotent stem cells, hiPSCs), which provides an effective means to study the mechanism of human early development,.HESCs and hiPSCs are known as human pluripotent stem cells (human pluripotent stem cells), with self renewal, unlimited proliferation and differentiation. The ability of a complete individual provides an effective means to study the development of cross germ cells, tissues and all adult cells. A large number of reports have been reported on the successful differentiation of three germ cells from hPSCs in vitro. The study on the induction of differentiation based on hPSCs to hematopoietic cells can greatly reappear in early human hematopoiesis. In order to study the development of MC and M in the early human development and to better simulate the early development of the human body, it is necessary to establish an in vitro culture system that tends to become a natural development process in vitro. We have studied the function and recognition ability of the innate immune cells produced by early hematopoietic development. Here, we take the initial development of human inherent immune cells as the research direction, and focus on exploring the origin and function of the initial development of MC and M diameter induced by hPSCs in vitro. Methods: Based on the hPSCs/ AGM-S3 co culture system, the inherent immune cell body is established. The morphology, phenotypic characteristics and functions of two kinds of innate immune cells were detected by MC and M diameter, and the origin of two kinds of cells was explored. The specific operation methods were as follows: 1. in mice aorta sex gland medium kidney (Aorta-Gonad-Mesonephros, AGM) derived matrix cell line (AGM-S3), co culture with hPSCs in vitro, induced to produce a large amount of production. Blood stem / progenitor cells, further suspension culture directed production of M and MC. culture methods were divided into three steps: 1) hPSCs cells were inoculated on AGM-S3 to co culture, induce hematopoietic differentiation, monitor the morphological and phenotypic expression changes at different time points, 2) after a week of suspension culture, amplification of hematopoietic stem / progenitor cells, 3) to replace the medium for MC or M The morphological and phenotypic characteristics of two kinds of innate immune cells were observed by.2., 1) MC morphological characteristics: mcgren Giemsa (May-GrunwaldGiemsa, MGG), acid toluidine blue (Acidictoluidineblue) and alnew blue (Alcian blue) chemical staining to observe cell morphology; electron microscope observation of MC particle morphology; immunofluorescence staining to observe Carboxy in MC Peptidase A, Cathepsin-G, Tryptase and Chymase protease expression, polychromatic flow detection technique analysis of MC phenotypic molecular expression lineage.2) M diameter morphological characteristics: MGG staining to observe the morphology of three sources M diameter; polychromatic flow detection technique analysis three sources M diameter phenotypic molecular table, two kinds of inherent immune cell function 1) Can detect: enzyme linked immunosorbent assay test IgE, Substance P and Compound48/80 stimulation, the content of histamine in MC supernatant and cell precipitation, RT-qPCR method to detect TLRs expression.2) function test: using RT-qPCR method to detect the expression of inflammatory factors and TLRs under different polarization conditions, and detect the phagocytic energy of M by low density lipoprotein (LDL) phagocytosis test. Force.4. to search for the progenitor cells of MC: using the flow sorting technique (Fluorescence Activated Cell Sorting, FACS) to select the target cell group and determine the result of MC progenitor cells: 1. a highly efficient hPSCs/AGM-S3 co culture hematopoietic cell differentiation system was established, and a large number of high purity MC and first M.1) were produced for 14 days and then cultured for 10 days. It was found that the MC of C-KIT+, CD45+, Tryptase+ and Chymase+, and the ratio of double yang to 98%.2 in 40 days was 3 days and then directed to M diameter for 14 days. The two natural immune cells produced at the initial stage of M may have tissue biased 1) chemical staining, immunofluorescence staining, electron microscopy and IgE, SubstanceP and Compound48/80 stimuli. Amines, determine that the MC produced by our culture system is MCTC type.2), and the initial production of M mm in IL-4 stimulation can be effectively polarized into M2 type 3., which is different from that of adult HSCs derived natural immune cells, 1) in peripheral blood mononuclear cells as control. Compared with MC from umbilical cord blood CD34+ cells, hESCs/AGM-S3 co culture is produced. The MC high expression of TLR2, TLR4 and TLR9.2) compared with the umbilical cord blood CD34+ cell source M diameter and the co culture of the 14 day source M diameter, the 3 days of co culture, M I phenotypic molecules have distinct expression levels of.IL-4 stimuli co cultured for 3 days and the TLR1 ~ TLR8 gene expression level in M is significantly higher than the two natural immune cells produced by the first two sources. Born before adult hematopoietic stem / progenitor cell (CD34+CD45+) 1) flow type separation co culture eighth days CD34+C-KIT- and CD34+C-KIT+ two groups of cells, directed to MC for 14 days, immunofluorescence staining showed that two subgroups produced progeny cells Tryptase and Chymase Shuangyang percentage were more than 85%.2) co cultured for third days of M a D, born in adult hematopoietic stem / hematopoietic stem. Prior to progenitor cells. Conclusion: the 1.hPSCs/AGM-S3 hematopoietic co culture system can induce the production of inherent immune cells such as MC and M diameter. And before the birth of adult hematopoietic stem / progenitor cells (CD34+CD45+ cells), a group of MC and M,.2., MC and M have their own unique developmental pathway,.3. these initial immunocytes are biased toward the tissue. The high expression of TLRs indicates that MC and M in tissue are stronger in immune recognition and play an important role in innate immune response.

【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】：2017
【分類號(hào)】：R392

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