本文選題：羅非魚　切入點：胚胎干細(xì)胞系　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：胚胎干(ES)細(xì)胞來源于早期胚胎囊胚的內(nèi)細(xì)胞團(tuán),具有自我更新與多向分化潛能,在再生醫(yī)學(xué)、發(fā)育生物學(xué)、功能基因組學(xué)等領(lǐng)域具有極大應(yīng)用前景,成為了當(dāng)前生命科學(xué)研究的重點和熱點課題。ES細(xì)胞分化的抑制是其體外培養(yǎng)的首要課題。自1981年小鼠胚胎干細(xì)胞首次成功建立以來,研究者相繼采用飼養(yǎng)細(xì)胞、含不同生長因子如白血病抑制因子(leukemia inhibitory factor,LIF)的條件培養(yǎng)基等抑制細(xì)胞的分化,但迄今僅少數(shù)物種如人、豬、牛、猴成功建立了ES細(xì)胞系。因此,ES細(xì)胞未分化狀態(tài)維持及其分子機(jī)制在不同物種中的保守性如何,尚待進(jìn)一步研究。魚類ES細(xì)胞的研究主要局限于小型模式魚類青溕(Oryzias latipes)和斑馬魚(Danio renio)。采用無飼養(yǎng)細(xì)胞的條件培養(yǎng)基(含魚類胚胎提取物、自身血清)目前已成功建立了青溕、斑馬魚ES細(xì)胞系,同時從一些海水魚中也得到了類似ES細(xì)胞的培養(yǎng)物。從而表明,飼養(yǎng)細(xì)胞對于魚類ES細(xì)胞系的建立并非必需。羅非魚(在本研究中,如未特殊說明均指尼羅羅非魚(Oreochromis niloticus))作為世界性養(yǎng)殖魚類,具有生長快、抗逆性強(qiáng)、繁殖周期短(14天)等特點,是開展ES細(xì)胞研究及其應(yīng)用研究的理想對象。LIF是IL-6(白細(xì)胞介素-6)家族重要成員,在造血干細(xì)胞、間充質(zhì)干細(xì)胞、原始生殖細(xì)胞、神經(jīng)干細(xì)胞的增殖、存活等方面均具有重要作用,特別是在ES細(xì)胞未分化狀態(tài)維持方面的作用,備受關(guān)注。大量研究表明,LIF通過激活JAK/STAT3、PI3K/AKT信號通路維持小鼠ES細(xì)胞的未分化狀態(tài),但其未能維持人ES細(xì)胞的未分化狀態(tài)。LIF在其他物種ES細(xì)胞中的作用如何,對于揭示ES細(xì)胞多能性調(diào)控機(jī)制的分子進(jìn)化具有積極作用。鑒于此,本研究以羅非魚為對象,開展了以下兩方面研究:一是采用條件培養(yǎng)基分離、培養(yǎng)羅非魚胚胎囊胚中期細(xì)胞,成功建立了胚胎干細(xì)胞系(命名為TES1),通過體外多能性分子檢測、體外分化誘導(dǎo)、胚胎嵌合體的形成等對其體內(nèi)外多能性及分化潛能進(jìn)行了鑒定,通過細(xì)胞增殖能力的檢測對其培養(yǎng)基中的不同成分進(jìn)行了檢測與優(yōu)化;二是對羅非魚Lif蛋白(OnLif)在TES1細(xì)胞的增殖、存活及未分化狀態(tài)的維持中的作用進(jìn)行了深入研究,主要研究結(jié)果如下:1.胚胎干細(xì)胞系的建立1.1細(xì)胞的分離、培養(yǎng)從羅非魚中期囊胚分離細(xì)胞,用含Hepes、青-鏈霉素、胎牛血清(FBS)、非蛋白因子5N(包括谷氨酰胺、丙酮酸鈉、亞硒酸鈉、非必需氨基酸和β-巰基乙醇)、人成纖維細(xì)胞生長因子(bFGF)、羅非魚胚胎提取物(TEE)及其血清(TS)的DMEM條件培養(yǎng)基在28℃培養(yǎng),獲得三個細(xì)胞系TES1-3,都表現(xiàn)出ES細(xì)胞樣的特征。本文以TES1為主要研究對象,在無飼養(yǎng)層培養(yǎng)條件下穩(wěn)定傳代至59代200天,仍具有ES細(xì)胞樣的克隆形成能力及典型的ES細(xì)胞樣表型特征(細(xì)胞圓形或多角形、核大、核仁明顯等)。1.2體外多能性堿性磷酸酶(AP)活性及多能性基因的表達(dá)是ES細(xì)胞檢測的重要指標(biāo)。在本研究中,AP活性檢測發(fā)現(xiàn),第55代TES1細(xì)胞均呈現(xiàn)強(qiáng)陽性;RT-PCR分析結(jié)果顯示,第16代及第50代TES1細(xì)胞均明顯表達(dá)pou5f3,sox2,myc和klf4等多能性基因,此外,通過熒光免疫組化實驗,從蛋白水平檢測到Pou5f3顯著表達(dá)于TES1細(xì)胞。這些結(jié)果證明,TES1細(xì)胞具有體外的多能性。1.3體外分化潛能證明ES細(xì)胞具有體外分化能力的重要手段是誘導(dǎo)細(xì)胞分化形成內(nèi)、中、外3個胚層,并最終得到擬胚體(EB)。本研究中,通過在培養(yǎng)基中加入誘導(dǎo)劑RA,TES1經(jīng)過10天懸浮培養(yǎng),形成類似于EB形態(tài)的細(xì)胞團(tuán)�，F(xiàn)有研究已證明nf200,actn2,hnf3b和sox10分別是內(nèi)、中、外3個胚層及神經(jīng)嵴特異的分化基因,RT-PCR分析證實,EB細(xì)胞團(tuán)均表達(dá)這些分化基因。同時在誘導(dǎo)后貼壁的細(xì)胞中也觀察到多種不同類型的分化細(xì)胞如星型細(xì)胞、神經(jīng)元細(xì)胞和扁平細(xì)胞等。由此表明,TES1具有體外多向分化的潛能。1.4體內(nèi)分化潛能將標(biāo)記了PKH26(活細(xì)胞紅色熒光染料)的TES1細(xì)胞移植進(jìn)入受體羅非魚中期囊胚,胚胎發(fā)育后期發(fā)現(xiàn)有35%的胚胎(n=501)是PKH26細(xì)胞嵌合體,有13%嵌合體胚胎發(fā)育成幼魚,同時觀察發(fā)現(xiàn)PKH26陽性細(xì)胞隨著胚胎的發(fā)育分布在羅非魚身體的不同部位,如軀干、眼和鰭,從而表明,TES1細(xì)胞在體內(nèi)具有多向分化潛能。1.5染色體分析對第40代TES1細(xì)胞的染色體經(jīng)分析發(fā)現(xiàn),在統(tǒng)計的100個細(xì)胞中,超過70%都是二倍體核型(2n=44),同時其形態(tài)未見異常。從而表明,長期培養(yǎng)的TES1細(xì)胞具有遺傳穩(wěn)定性。1.6不同成分對TES1細(xì)胞增殖活性的影響為優(yōu)化培養(yǎng)條件,TES1細(xì)胞正常傳代培養(yǎng)至第28代時,通過CCK-8分別檢測多種因子對其增殖的影響。結(jié)果顯示,FBS濃度顯著影響細(xì)胞增殖活性,在0-15%濃度范圍內(nèi),FBS濃度越高,細(xì)胞增殖活性越強(qiáng),超過15%時,隨著FBS濃度增高細(xì)胞增殖活性越弱,表明TES1的增殖具有FBS濃度依賴;培養(yǎng)基中其他成分,包括胚胎提取物、魚血清、5N均能促進(jìn)細(xì)胞增殖,其中自身胚胎提取物及自身血清的促增殖活性顯著高于其他魚類來源的胚胎提取物及血清,從而表明,其促增殖活性既有物種保守性同時具有一定的物種特異性;不同濃度(5,10,20 ng/ml)bFGF均能促進(jìn)細(xì)胞增殖,其中10ng/ml是其最適濃度。2.OnLif對TES1未分化狀態(tài)的維持2.1細(xì)胞增殖用分別含有1、10、100 ng/ml OnLif的基礎(chǔ)培養(yǎng)基處理TES1細(xì)胞24、48、72、96 h后,CCK-8檢測結(jié)果顯示,1、100 ng/ml OnLif對TES1的增殖無明顯促進(jìn)作用(p0.05),而10 ng/ml OnLif從48 h開始能顯著促進(jìn)TES1的增殖(p0.01);EdU摻入法檢測進(jìn)一步證實了上述結(jié)果,從而表明,OnLif能明顯促進(jìn)TES1的增殖并且具有濃度依賴性。2.2細(xì)胞存活在低、中、高細(xì)胞密度條件下,用基礎(chǔ)培養(yǎng)基、含10 ng/ml OnLif基礎(chǔ)培養(yǎng)基及完全培養(yǎng)基TESM處理TES1,結(jié)果發(fā)現(xiàn),在低、高密度條件下,對照組大量細(xì)胞凋亡,特別是低密度條件下幾乎無細(xì)胞存活,而含On Lif的實驗組與TESM組細(xì)胞狀態(tài)基本一致,細(xì)胞生長狀態(tài)良好,未觀察到明顯細(xì)胞凋亡;在中密度條件下,對照組光鏡下雖然未觀察到明顯的細(xì)胞凋亡,AnnexinV-FITC/PI細(xì)胞凋亡雙染實驗結(jié)果發(fā)現(xiàn),24 h對照組與實驗組無顯著差異,但是48 h對照組細(xì)胞凋亡率(22.8%)明顯高于實驗組(8.9%)(p0.01)。從而表明,OnLif能抑制TES1細(xì)胞的凋亡,促進(jìn)細(xì)胞存活。2.3多能性活性用含OnLif培養(yǎng)基培養(yǎng)TES1細(xì)胞3天和5天后,半定量RT-PCR和AP活性檢測細(xì)胞多能性活性,結(jié)果顯示,實驗組細(xì)胞多能性標(biāo)志基因pou5f3、sox2、myc、klf4的表達(dá)及AP活性明顯高于對照組,分化標(biāo)志基因nf200、actn2、hnf3b的表達(dá)低于對照組或不表達(dá),此外,Pou5f3的免疫組化及其啟動子載體pT2AL-Onpou5f3-GFP轉(zhuǎn)染進(jìn)一步證實實驗組細(xì)胞多能性活性明顯強(qiáng)于對照組。這些表明,OnLif可以促進(jìn)TES1細(xì)胞的未分化狀態(tài)的維持。本研究成功建立了羅非魚胚胎干細(xì)胞系,并首次證實白血病抑制因子在維持魚類胚胎干細(xì)胞未分化狀態(tài)中的作用,將推進(jìn)我們對LIF在魚類ES細(xì)胞自我更新和維持中作用機(jī)制的進(jìn)一步認(rèn)識,對揭示ES細(xì)胞多能性調(diào)控機(jī)制的分子進(jìn)化具有積極作用。
[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.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R329.2

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