人多能干细胞体外诱导产生肥大和巨噬细胞的初期发育及功能研究

发布时间：2018-05-10 15:58

本文选题：人类多潜能干细胞(hPSCs) + AGM-S3　；参考：《北京协和医学院》2017年博士论文

【摘要】：研究背景和目的:肥大细胞(Mast cells,MC)和巨噬细胞(Macrophage,MΦ)是非常重要的免疫细胞,在机体固有免疫反应中具有举足轻重的作用。MC和MΦ具有极大的异质性。在人类MΦ既有分布于血液中以单核形式存在的状态,也有在多种组织和器官中分布的组织驻留型,并根据分布的形态和功能具有不同命名;而人类MC根据颗粒中含有蛋白酶种类不同,分为MCT和MCTc两类,前者与小鼠黏膜MC相似,后者与小鼠组织相关MC类似。MΦ和MC表面和内体分布多种Toll样受体(TLRs,一种机体内重要的模式识别受体),可有效识别病原体相关外源性危险信号(PAMPs)和自身细胞所释放的内源性危险信号(DAMPs),在机体防御中发挥着重要的作用。在个体发生的早期阶段,血细胞起源在顺序上有着时空分布的不同特征。所有血细胞均来源于中胚层,最早的造血细胞起源于卵黄囊;随后进入胚内的主动脉-性腺-中肾区域(AGM区),在此可产生最初的能够确认的造血干细胞;最后进入胎肝造血,在此含有大量可移植的造血干细胞。依据人体造血发生的时空顺序及不同特征,可将其分为原始造血和成体造血。在成体造血,所有功能型血细胞均来源于骨髓中的造血干细胞(Hematopoietic stem cells,HSCs)。然而,对小鼠胚胎造血发育的研究显示,起源于卵黄囊的原始造血以及红系/髓系祖细胞(EMP)都可产生MΦ。另外,在小鼠胎肝发育过程中发现MC前体高度集中于卵黄囊和胎肝血中,提示MΦ和MC存在一个较强的早期胚性发育阶段。研究发现,产生于早期胚性发育阶段的这两种固有免疫细胞具有组织偏向特性。由于受伦理道德及法律的限制,在人体中进行胚胎造血发育的研究存在诸多困难,导致对人类早期胚胎造血发生认识的缺失。对造血细胞发生学的研究主要基于小鼠等动物模型。但是动物体内的研究数据并不能完全指示人体内的发育过程。随着人胚胎干细胞(human embryonic stem cells,hESCs)的成功建系以及人诱导性多能干细胞(human induced pluripotent stem cells,hiPSCs)的体外重编程获得,为研究人类早期发育机制提供了有效的手段。hESCs和hiPSCs统称为人多能干细胞(human pluripotent stem cells,hPSCs),具有自我更新、无限增殖及分化形成完整个体的能力,为研究跨胚层细胞、组织以及所有成体细胞的发育,提供了有效的手段。已有大量关于从hPSCs体外成功分化出三个胚层来源成熟细胞的报道。基于hPSCs向造血细胞诱导分化的研究,可很大程度上重现早期人类造血发生过程,有助于详尽地解析人类胚胎期造血的发育模式。为了研究人类早期发育过程中MC和MΦ的发育情况,从而更好地模拟体内早期发育,需要在体外建立一种高效地趋于自然发育过程的培养体系。通过与成体HSCs来源固有免疫细胞比较,我们研究了早期造血发育产生固有免疫细胞的功能及识别能力。在此,我们以人固有免疫细胞初期发育作为研究方向,重点探索hPSCs体外诱导产生MC和MΦ初期发育的起源及其功能。研究方法:基于hPSCs/AGM-S3共培养体系,建立固有免疫细胞体外培养方法。以MC和MΦ为主,分别检测两种固有免疫细胞的形态、表型特征以及功能,并探索两种细胞的起源。具体操作方法如下:1.以小鼠主动脉-性腺-中肾(Aorta-Gonad-Mesonephros,AGM)来源基质细胞系(AGM-S3),与hPSCs体外共培养,诱导产生大量造血干/祖细胞,进一步悬浮培养定向产生MΦ和MC。培养方法分为三步:1)将hPSCs细胞接种到AGM-S3上进行共培养,诱导造血分化发生,监测不同时间点克隆形态及表型分子表达变化,2)经悬浮培养一周,扩增造血干/祖细胞,3)更换培养基进行MC或MΦ定向分化。2.观察两种固有免疫细胞形态及表型特征1)MC形态学特征:麦格氏-吉姆萨(May-GrunwaldGiemsa,MGG)、酸性甲苯胺蓝(Acidictoluidineblue)以及阿尔新蓝(Alcian blue)化学染色观察细胞形态;电镜观察MC内颗粒形态;免疫荧光染色观察MC中Carboxypeptidase A、Cathepsin-G、Tryptase和Chymase蛋白酶的表达情况,多色流式检测技术分析MC表型分子表达谱系。2)MΦ形态学特征:MGG染色观察三种来源MΦ形态;多色流式检测技术分析三种来源MΦ表型分子表达谱系。3.检测两种固有免疫细胞功能1)MC功能检测:酶联免疫吸附法检测IgE、Substance P和Compound48/80刺激下,MC上清及细胞沉淀中组胺的含量;RT-qPCR方法检测TLRs表达。2)Φ功能检测:利用RT-qPCR方法检测不同极化条件下,炎症因子及TLRs表达;通过低密度脂蛋白(LDL)吞噬实验检测MΦ吞噬能力。4.寻找MC的祖细胞:利用流式分选技术(Fluorescence Activated Cell Sorting,FACS)分选目标细胞群,确定MC祖细胞结果:1.建立高效的hPSCs/AGM-S3共培养造血细胞诱导分化体系,可产生大量高纯度MC和初期MΦ。1)共培养14天再向MC定向培养10天,体系中发现C-KIT+,CD45+,Tryptase+和Chymase+的MC,40天双阳比例高达98%。2)共培养3天再向MΦ定向培养14天,可产生初期MΦ。2.产生的两种固有免疫细胞具有组织偏向型1)化学染色,免疫荧光染色,电镜观察以及IgE、SubstanceP和Compound48/80刺激下,MC都可释放组胺,确定我们的培养体系产生的MC为MCTC类型。2)初期产生MΦ在IL-4刺激可有效极化成M2型3.产生的两种固有免疫细胞与成体HSCs来源固有免疫细胞不同1)以外周血单个核细胞作为对照,与脐带血CD34+细胞来源的MC相比,hESCs/AGM-S3共培养产生的MC高表达TLR2,TLR4和TLR9。2)与脐带血CD34+细胞来源MΦ以及共培养14天来源MΦ相比,共培养3天来源MΦ表型分子具有明显不同的表达水平。IL-4刺激共培养3天来源MΦ中TLR1～TLR8基因表达水平明显高于前两种来源MΦ。4.产生的两种固有免疫细胞诞生于成体造血干/祖细胞(CD34+CD45+)之前1)流式分选共培养第8天CD34+C-KIT-和CD34+C-KIT+两群细胞,向MC定向培养14天,免疫荧光染色显示两亚群产生子代细胞Tryptase和Chymase双阳百分比都大于85%。2)共培养第3天产生的MΦD,诞生于成体造血干/祖细胞之前。结论:1.hPSCs/AGM-S3造血共培养体系可诱导产生MC和MΦ等固有免疫细胞,并且在成体造血干/祖细胞(CD34+CD45+细胞)诞生之前,可产生一群MC和MΦ。2.这群MC和MΦ具有自己独特的发育途径。3.这些初期产生的固有免疫细胞偏向于组织特性,高表达TLRs,说明分布于组织中的MC和MΦ具有更强的免疫识别能力,在固有免疫反应中发挥着重要的作用。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R392

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