尼罗罗非鱼胚胎干细胞系的建立及白血病抑制因子维持其未分化状态的研究

发布时间：2018-03-19 02:19

本文选题：罗非鱼　切入点：胚胎干细胞系　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：胚胎干(ES)细胞来源于早期胚胎囊胚的内细胞团,具有自我更新与多向分化潜能,在再生医学、发育生物学、功能基因组学等领域具有极大应用前景,成为了当前生命科学研究的重点和热点课题。ES细胞分化的抑制是其体外培养的首要课题。自1981年小鼠胚胎干细胞首次成功建立以来,研究者相继采用饲养细胞、含不同生长因子如白血病抑制因子(leukemia inhibitory factor,LIF)的条件培养基等抑制细胞的分化,但迄今仅少数物种如人、猪、牛、猴成功建立了ES细胞系。因此,ES细胞未分化状态维持及其分子机制在不同物种中的保守性如何,尚待进一步研究。鱼类ES细胞的研究主要局限于小型模式鱼类青溕(Oryzias latipes)和斑马鱼(Danio renio)。采用无饲养细胞的条件培养基(含鱼类胚胎提取物、自身血清)目前已成功建立了青溕、斑马鱼ES细胞系,同时从一些海水鱼中也得到了类似ES细胞的培养物。从而表明,饲养细胞对于鱼类ES细胞系的建立并非必需。罗非鱼(在本研究中,如未特殊说明均指尼罗罗非鱼(Oreochromis niloticus))作为世界性养殖鱼类,具有生长快、抗逆性强、繁殖周期短(14天)等特点,是开展ES细胞研究及其应用研究的理想对象。LIF是IL-6(白细胞介素-6)家族重要成员,在造血干细胞、间充质干细胞、原始生殖细胞、神经干细胞的增殖、存活等方面均具有重要作用,特别是在ES细胞未分化状态维持方面的作用,备受关注。大量研究表明,LIF通过激活JAK/STAT3、PI3K/AKT信号通路维持小鼠ES细胞的未分化状态,但其未能维持人ES细胞的未分化状态。LIF在其他物种ES细胞中的作用如何,对于揭示ES细胞多能性调控机制的分子进化具有积极作用。鉴于此,本研究以罗非鱼为对象,开展了以下两方面研究:一是采用条件培养基分离、培养罗非鱼胚胎囊胚中期细胞,成功建立了胚胎干细胞系(命名为TES1),通过体外多能性分子检测、体外分化诱导、胚胎嵌合体的形成等对其体内外多能性及分化潜能进行了鉴定,通过细胞增殖能力的检测对其培养基中的不同成分进行了检测与优化;二是对罗非鱼Lif蛋白(OnLif)在TES1细胞的增殖、存活及未分化状态的维持中的作用进行了深入研究,主要研究结果如下:1.胚胎干细胞系的建立1.1细胞的分离、培养从罗非鱼中期囊胚分离细胞,用含Hepes、青-链霉素、胎牛血清(FBS)、非蛋白因子5N(包括谷氨酰胺、丙酮酸钠、亚硒酸钠、非必需氨基酸和β-巯基乙醇)、人成纤维细胞生长因子(bFGF)、罗非鱼胚胎提取物(TEE)及其血清(TS)的DMEM条件培养基在28℃培养,获得三个细胞系TES1-3,都表现出ES细胞样的特征。本文以TES1为主要研究对象,在无饲养层培养条件下稳定传代至59代200天,仍具有ES细胞样的克隆形成能力及典型的ES细胞样表型特征(细胞圆形或多角形、核大、核仁明显等)。1.2体外多能性碱性磷酸酶(AP)活性及多能性基因的表达是ES细胞检测的重要指标。在本研究中,AP活性检测发现,第55代TES1细胞均呈现强阳性;RT-PCR分析结果显示,第16代及第50代TES1细胞均明显表达pou5f3,sox2,myc和klf4等多能性基因,此外,通过荧光免疫组化实验,从蛋白水平检测到Pou5f3显著表达于TES1细胞。这些结果证明,TES1细胞具有体外的多能性。1.3体外分化潜能证明ES细胞具有体外分化能力的重要手段是诱导细胞分化形成内、中、外3个胚层,并最终得到拟胚体(EB)。本研究中,通过在培养基中加入诱导剂RA,TES1经过10天悬浮培养,形成类似于EB形态的细胞团。现有研究已证明nf200,actn2,hnf3b和sox10分别是内、中、外3个胚层及神经嵴特异的分化基因,RT-PCR分析证实,EB细胞团均表达这些分化基因。同时在诱导后贴壁的细胞中也观察到多种不同类型的分化细胞如星型细胞、神经元细胞和扁平细胞等。由此表明,TES1具有体外多向分化的潜能。1.4体内分化潜能将标记了PKH26(活细胞红色荧光染料)的TES1细胞移植进入受体罗非鱼中期囊胚,胚胎发育后期发现有35%的胚胎(n=501)是PKH26细胞嵌合体,有13%嵌合体胚胎发育成幼鱼,同时观察发现PKH26阳性细胞随着胚胎的发育分布在罗非鱼身体的不同部位,如躯干、眼和鳍,从而表明,TES1细胞在体内具有多向分化潜能。1.5染色体分析对第40代TES1细胞的染色体经分析发现,在统计的100个细胞中,超过70%都是二倍体核型(2n=44),同时其形态未见异常。从而表明,长期培养的TES1细胞具有遗传稳定性。1.6不同成分对TES1细胞增殖活性的影响为优化培养条件,TES1细胞正常传代培养至第28代时,通过CCK-8分别检测多种因子对其增殖的影响。结果显示,FBS浓度显著影响细胞增殖活性,在0-15%浓度范围内,FBS浓度越高,细胞增殖活性越强,超过15%时,随着FBS浓度增高细胞增殖活性越弱,表明TES1的增殖具有FBS浓度依赖;培养基中其他成分,包括胚胎提取物、鱼血清、5N均能促进细胞增殖,其中自身胚胎提取物及自身血清的促增殖活性显著高于其他鱼类来源的胚胎提取物及血清,从而表明,其促增殖活性既有物种保守性同时具有一定的物种特异性;不同浓度(5,10,20 ng/ml)bFGF均能促进细胞增殖,其中10ng/ml是其最适浓度。2.OnLif对TES1未分化状态的维持2.1细胞增殖用分别含有1、10、100 ng/ml OnLif的基础培养基处理TES1细胞24、48、72、96 h后,CCK-8检测结果显示,1、100 ng/ml OnLif对TES1的增殖无明显促进作用(p0.05),而10 ng/ml OnLif从48 h开始能显著促进TES1的增殖(p0.01);EdU掺入法检测进一步证实了上述结果,从而表明,OnLif能明显促进TES1的增殖并且具有浓度依赖性。2.2细胞存活在低、中、高细胞密度条件下,用基础培养基、含10 ng/ml OnLif基础培养基及完全培养基TESM处理TES1,结果发现,在低、高密度条件下,对照组大量细胞凋亡,特别是低密度条件下几乎无细胞存活,而含On Lif的实验组与TESM组细胞状态基本一致,细胞生长状态良好,未观察到明显细胞凋亡;在中密度条件下,对照组光镜下虽然未观察到明显的细胞凋亡,AnnexinV-FITC/PI细胞凋亡双染实验结果发现,24 h对照组与实验组无显著差异,但是48 h对照组细胞凋亡率(22.8%)明显高于实验组(8.9%)(p0.01)。从而表明,OnLif能抑制TES1细胞的凋亡,促进细胞存活。2.3多能性活性用含OnLif培养基培养TES1细胞3天和5天后,半定量RT-PCR和AP活性检测细胞多能性活性,结果显示,实验组细胞多能性标志基因pou5f3、sox2、myc、klf4的表达及AP活性明显高于对照组,分化标志基因nf200、actn2、hnf3b的表达低于对照组或不表达,此外,Pou5f3的免疫组化及其启动子载体pT2AL-Onpou5f3-GFP转染进一步证实实验组细胞多能性活性明显强于对照组。这些表明,OnLif可以促进TES1细胞的未分化状态的维持。本研究成功建立了罗非鱼胚胎干细胞系,并首次证实白血病抑制因子在维持鱼类胚胎干细胞未分化状态中的作用,将推进我们对LIF在鱼类ES细胞自我更新和维持中作用机制的进一步认识,对揭示ES细胞多能性调控机制的分子进化具有积极作用。
[Abstract]:Embryonic stem (ES) cells derived from inner cell mass of the blastocyst embryos, with self-renewal and multipotential differentiation, developmental biology in regenerative medicine, and has great application prospect in functional genomics and other fields, has become the focus of life science research hot topic and differentiation of.ES cells inhibited the in vitro culture is the first topic since 1981. The first successful establishment of mouse embryonic stem cells, researchers have adopted the feeder cells, containing different growth factors such as leukemia inhibitory factor (leukemia inhibitory, factor, LIF) of the base cell differentiation inhibited culture conditions, but so far only a few species such as human, pig, ox, monkey successfully established ES cell line therefore, how to maintain the undifferentiated state of ES cells and its molecular mechanism in different species conservation, still needs further research. The main research is limited to small fish ES cell model of fish Green Meng (Oryzias latipes) and zebrafish (Danio renio). The culture medium without feeder cells conditions (including fish embryo extract, its serum) has successfully established the green Meng, zebrafish ES cell line, and from some marine fish were obtained in cultures similar to ES cells. Thus indicating that feeder cells the fish ES cell line establishment is not necessary. Tilapia (in this study, if no special instructions are refers to the Nile tilapia (Oreochromis niloticus)) as the world fish, with fast growth, strong resistance, breeding period (14 days) and other characteristics, is an ideal object to carry out research on ES cell research and.LIF the application of IL-6 (interleukin -6) is an important member in the family of hematopoietic stem cells, mesenchymal stem cells, primordial germ cells, the proliferation of neural stem cells, which plays an important role in survival, especially in undifferentiated ES cells. To maintain the state of the role of concern. Many studies indicate that LIF through activating JAK/STAT3 and PI3K/AKT signaling pathway in mouse ES cells maintain undifferentiated state, but it failed to maintain ES cells in an undifferentiated state how the role of.LIF in other species in ES cells, and plays a positive role in revealing the molecular evolution of ES cells can the mechanism of regulation. In view of this, this study using tilapia as the object, to carry out the following two aspects: one is using conditioned medium separation, cultured tilapia blastocyst metaphase cells, successfully established embryonic stem cell line (named TES1), through in vitro pluripotency molecular detection, in vitro differentiation, embryo block fit the formation of in vivo in vitro pluripotency and differentiation were identified by detection and optimization of the culture medium of different components was detected in cell proliferation; two of tilapia L If protein (OnLif) in TES1 cell proliferation, survival and markerexpression role of in-depth research, the main results are as follows: 1. the establishment of embryonic stem cell lines isolated from 1.1 cells, cultured tilapia from the mid blastula cells were isolated, with Hepes, penicillin streptomycin, fetal bovine serum (FBS), non protein factor 5N (including glutamine, sodium pyruvate, sodium selenite, non essential amino acids and beta mercaptoethanol), fibroblast growth factor (bFGF), tilapia embryo extract (TEE) and serum DMEM (TS) a train at 28 C medium, three cell lines TES1-3, showed the characteristics of ES cell samples. This paper takes TES1 as the main research object in feeder free culture conditions of stable passage to the 59 generation of 200 days, still has a kind of ES cell clone formation ability and the typical ES cell like characteristics (table cell nucleus round or polygonal, Nucleolus etc.).1.2 in vitro to alkaline phosphatase (AP) activity and expression of pluripotency genes is an important index to detect ES cells. In this study, found that AP activity detection, the fifty-fifth generation of TES1 cells showed strong positive; RT-PCR analysis showed that the sixteenth generation and the 50 generation of TES1 cells were observed in the expression of pou5f3, Sox2, myc and KLF4 pluripotency genes, in addition, by fluorescence immunohistochemical experiments, from the protein level detected Pou5f3 expression significantly in TES1 cells. These results suggested that TES1 cells with in vitro pluripotent differentiation potential in vitro.1.3 proved an important means of ES cells with in vitro differentiation ability is the induction of cell differentiation the formation, in 3 layers, and finally obtain embryoid bodies (EB). In this study, through the medium with inducer RA, TES1 after 10 days of suspension culture, the formation of similar EB form cell clusters. Existing studies have demonstrated that NF200, AC Tn2, hnf3b and Sox10 respectively, and differentiation of gene 3 embryonic and neural crest specific, RT-PCR analysis confirmed that the gene was expressed in EB cells differentiation. At the same time in adherent cells was also observed in a variety of different types of differentiated cells such as astrocytes, nerve cells and flat element cell. This shows that the potential in vivo differentiation potential of.1.4 TES1 has in vitro differentiation marker PKH26 (live cells red fluorescent dye) TES1 cells transplanted into the receptor of tilapia mid blastula embryos later found 35% embryos (n=501) is a PKH26 cell chimerism, a chimera embryos juvenile block 13%. At the same time we found that PKH26 positive cells during embryogenesis are distributed in different parts of the body such as the trunk, tilapia, eye and fins, which showed that TES1 cells have multilineage differentiation potential of.1.5 chromosome analysis of fortieth in vivo Generation of TES1 cells by chromosome analysis showed that in the 100 cell statistics, more than 70% are diploid karyotype (2n=44), and no abnormal shape. It shows that the long-term cultured TES1 cells have the genetic stability of different components of.1.6 on TES1 cell proliferation effect in order to optimize the culture conditions, TES1 cells were normal cultured to the twenty-eighth generation, influence of various factors on the proliferation were detected by CCK-8. The results showed that FBS concentration significantly affect cell proliferation activity in the concentration range of 0-15%, the higher the concentration of FBS, cell proliferation activity is stronger, more than 15%, with the concentration of FBS increased cell proliferation activity is weak, showed that the proliferation of TES1 with FBS concentration dependence; other medium components, including embryo extract, fish serum, 5N could promote cell proliferation, including its embryo extract and its serum proliferative activity was significantly higher than that of other fish Embryo extract and serum, which indicates that the source of the class, proliferative activity of both species conservation and species specificity of certain; different concentrations (5,10,20 ng/ml) bFGF could promote cell proliferation, in which 10ng/ml is the most suitable concentration of.2.OnLif containing 1,10100 ng/ml OnLif respectively based on TES1 markerexpression 2.1 cell proliferation medium TES1 cells treated with 24,48,72,96 after h, CCK-8 test results showed that 1100 ng/ml OnLif on the proliferation of TES1 no obvious effect (P0.05), and 10 ng/ml OnLif from 48 h could significantly promote the proliferation of TES1 (P0.01); EdU incorporation assay further confirmed the results, which show that OnLif can obviously promote the proliferation of TES1 and.2.2 in a concentration dependent cell survival in low, in conditions of high cell density, medium, containing 10 ng/ml OnLif basic culture medium and culture medium T ESM TES1, found that in low, high density condition, the control group and apoptosis, especially under the condition of low density almost no cell survival, while containing On Lif experimental group and TESM group cells consistent with cell growth in good condition, there were no obvious apoptosis; in the condition of density the control group under light microscope, while not observed significant cell apoptosis, apoptosis of AnnexinV-FITC/PI double staining experiment results showed that the 24 h control group had no significant difference with the experimental group, but the 48 h control group apoptosis rate (22.8%) was significantly higher than the experimental group (8.9%) (P0.01). It showed that OnLif can inhibit apoptosis TES1 cells,.2.3 can promote cell survival TES1 cells cultured in OnLif medium containing 3 and 5 days of activity, cells were detected by semi quantitative RT-PCR and AP activity to activity, results show that the experimental group cell pluripotency markers pou5f3, Sox2, myc, The expression of KLF4 and AP activity was significantly higher than the control group, differentiation marker gene NF200, actn2, hnf3b expression was lower than that of the control group or no expression, in addition, the immunohistochemical Pou5f3 and promoter vector pT2AL-Onpou5f3-GFP was further confirmed in experimental group cell pluripotency activity was significantly stronger than the control group. These indicate that OnLif can promote the maintenance of TES1 cells in an undifferentiated state. This study successfully established tilapia embryonic stem cell lines, and the first time that leukemia inhibitory factor in fish cells maintain undifferentiated state of embryonic stem, will promote the further understanding of LIF in fish ES cells to self renew and maintain the mechanism of us, to reveal the molecular ES cells the evolution of the regulatory mechanism has a positive effect.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2

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