gfi1基因调控斑马鱼造血发育的机理研究

发布时间：2018-05-15 06:03

本文选题：CRISPR-Cas9 + TALEN　；参考：《南方医科大学》2016年硕士论文

【摘要】：研究背景血液系统疾病(如地中海贫血、免疫缺陷和白血病等)一直是影响我国人民健康的严重疾病,其发病率居高不下给整个社会的医疗保障体系带来沉重负担。究其深层次的原因,血液疾病发病率居高不下反映了人类对于造血调控和相关疾病发生的分子机制和病理机制认识不足,因为血液系统正确作用与维持稳态依赖于各种血细胞的正常发育,而造血过程缺陷会导致多种血细胞或血管发育障碍。因此,在分子和细胞水平更好地了解造血细胞的发育与造血过程,将有助于逐步明晰血液系统疾病发病机理,从而拓展临床治疗血液系统疾病的应用潜能。在脊椎动物中,造血系统发育过程是各种血细胞发育、成熟,同时受到多个因子调控的复杂而又有序的动态过程。尽管各种血细胞组分的生理功能不同,但是它们有着同一祖先——造血干细胞(hematopoietic stem cells,HSCs)。造血系统的发育中,造血干细胞早期发育是最核心的过程。HSC的正确产生、维持、增殖以及顺利迁移及定位,对于动物体的胚胎期的器官发生以及成体中组织稳态的维持以及损伤修复均必不可缺[1,2]。从HSC向成熟血细胞分化的过程中,经历了造血祖细胞阶段和造血前体细胞阶段,最终分化生成所有谱系成熟血细胞。造血干细胞向各谱系发育分化的过程中需要多种调节分子的参与,转录因子在此过程中起了重要的调控作用,并且转录因子之间发生相互调控,从而形成复杂的调控网络。转录因子在造血细胞发育分化过程中起着重要的作用,各转录因子在细胞发育的不同时间及空间表达增强或减弱,使得造血干细胞或祖细胞向着某一谱系分化。Gfi1作为一种参与造血调控的转录抑制因子,该基因缺失或突变的病人表现为粒细胞成熟缺陷,阻滞于早幼粒阶段。虽然在Gfi1基因敲除小鼠实验中未发生白血病,但是由于GFI1功能的缺失,有助于白血病的发生,特别是髓系细胞白血病的发生,如AML。经典的模式生物如线虫、果蝇等与人类同源性相差甚远,而传统模式动物小鼠等哺乳动物繁殖速度较慢,且体积相对较大,难以实施高通量造血缺陷突变体的筛选。而且哺乳动物为子宫内发育,不利于观察胚胎期造血的表型。斑马鱼作为脊椎动物,在器官发育、造血调控、疾病发生等方面与人类高度相似。特别地,斑马鱼的血液和心血管系统早期发育与人类极为相似,不论是血液成分的组成,还是发育过程都非常相似,而且该系统的发育缺陷突变体仍然可以存活数天,为人们研究胚胎早期造血调控提供了极为有利的条件[3]。本论文前一部分分别应用TALEN靶向敲除技术、CRISPR-Cas9敲除技术,成功对斑马鱼AB品系gfi1aa基因、gfi1b基因进行敲除,获得多种不同基因型的gfi1aako突变体、gfi1bko突变体。并详细研究突变体在造血过程(原始造血和永久造血)中各个谱系标记物的时空表达情况以及各个谱系细胞的发育情况,旨在探讨gfi1aa基因以及gfi1b基因调控红系造血发育的机理。论文后一部分主要探索a-catenin、β-catenin对胚胎期造血干细胞迁移的作用,以期进一步明确造血干细胞迁移过程的相关细胞和分子调控机制。论文内容共分为两个部分:第一部分参加全国"斑马鱼1号染色体全基因敲除"项目及gfi1aa调控斑马鱼造血发育的机理研究;第二部分a-catenin、β-catenin对胚胎期造血干细胞迁移的作用研究。第一部分参加全国"斑马鱼1号染色体全基因敲除"项目及gfi1基因调控斑马鱼造血发育的机制研究1.目的本部分首先介绍CRISPR-Cas9及TALEN基因编辑技术,然后以斑马鱼为模式生物,利用这两种技术完成了以下两项工作:1)利用CRISPR-Cas9技术,参与全国"斑马鱼1号染色体全基因敲除"项目,对1号染色体上的ZKO编号为863～873的11个基因(vps37c、tmem132a、dnaja1、smu1b、rps6、btr01、c1h20orf27、muc13a、si:ch211-239f4.6、mucms1、si:ch211-239f4.4)进行基因敲除,筛选稳定遗传的F2代突变体。2)通过TALEN靶向基因敲除技术,利用斑马鱼模式生物对其造血系统发育基因gfi1aa进行敲除,获得造血发育缺陷的gfi1aa突变体。同时利用CRISPR-Cas9敲除技术,对gfi1家族另一个gfi1b基因进行敲除并获得gfi1bko突变体。最后,利用这两个突变体进行表型与功能研究,探索gfi1基因调控造血发育的细胞以及分子机理。2.方法1)主要采用CRISPR-Cas9基因组编辑系统,利用gRNA的靶向识别及Cas9的核酸切割作用。通过NCBI、Ensemb1和Smart网站查找相关基因的基本信息,对ZKO编号为863～873的11个基因进行靶点设计,合成相应的引物。体外转录得到相应的gRNA和Cas9 mRNA,混合共同显微注射到one-cell的野生型AB斑马鱼胚胎中,得到F0代嵌合体的斑马鱼,将养大的FO(Founder)与野生型AB成鱼外交得到F1 embryos,通过对F1代胚胎进行PCR、酶切、测序的方法筛选出生殖细胞中带有靶位点突变的F0成鱼。将这样的F0外交得到的F1养大,再通过剪尾逐条检测F1的靶位点序列筛选可稳定遗传的斑马鱼个体。2)分别应用TALEN、CRISPR-Cas9基因敲除技术,对斑马鱼AB品系造血发育相关基因gfi1aa基因、gfi1b基因进行敲除,以期获得gfi1aako突变体、gfi1bko突变体。然后针对突变体在造血过程(原始造血和永久造血)中各个谱系标记物的时空表达情况以及各个谱系细胞的发育情况,探讨gfi1aa基因以及gfi1b基因调控造血发育的机理。3.结果1)利用Cas9基因敲除技术,得到单种基因型突变的F1代斑马鱼,然后再与AB外交得到F2代,通过PCR、酶切、测序的方法得到F2代杂合突变体斑马鱼,并将部分上交到国家斑马鱼资源中心。然后对剩下的F2代突变体斑马鱼进行intercross,通过原位杂交、苏丹黑B染色、中性红染色等方法进行表型筛选。2)利用TALEN靶向基因敲除技术对斑马鱼gfi1aa进行敲除,得到缺失23个碱基(5'-TCTTCTCAACAGTCCCCGCTGAG-3')的gfi1aako 突变体。表型分析结果显示:在斑马鱼造血发育早期,gfi1aa影响红细胞和中性粒细胞发育,而不影响巨噬细胞发育。而gfi1aa影响早期红系发育的细胞学机理主要是通过抑制红细胞的增殖,而并不影响红细胞前体细胞和红细胞的细胞形态。在50 hpf时期,gfi1aako纯合突变体血红蛋白表达量恢复到与野生型相当的水平。gfi1b是gfi1aa的同源基因,也高表达于红系及巨核系细胞,为了验证在红细胞发育的定向造血阶段是受gfi1b的调控,我们利用CRISPR-Cas9基因敲除技术获得gfi1bko的突变体斑马鱼,通过双突变体的实验结果发现在定向造血阶段,gfi1的家族基因gfi1b会补偿gfi1aa基因的功能。第二部分a-cateninn、β-catenin对胚胎期造血干细胞迁移的作用研究1.目的造血干细胞(HSC)发育缺陷会引起各种血细胞或血管发育障碍,并与很多血液系统疾病密切相关。而胚胎期造血干细胞的迁移是HSC正确发育的必经过程,然而人们对于胚胎发育期HSC迁移的机制知之甚少。目前与胚胎期造血干细胞迁移调控相关的分子研究较少,他们是黏附分子、细胞因子和趋化因子[4]。而β-catenin作为细胞内骨架的一种功能性蛋白,能和a-catenin及γ-catenin结合到黏附分子VE-cadherin上,作为蛋白复合体参与细胞间的黏附[5]。Takahashi等研究发现,恶性黑色素瘤B16细胞沉默β-catenin基因后促进肿瘤细胞迁移[6]。而前期我们对cMyb在胚胎期HSC迁移调控的报道[7],是cmyb基因功能研究中前所未有的发现。本部分我们以cmybhk=3斑马鱼缺陷突变体为模型,深入研究斑马鱼模型的a-catenin、β-catenin对于胚胎期HSC迁移及定位的机理,逐步完善胚胎期HSC发育调控的分子信号调控网络。2.方法首先,为了检测斑马鱼中a-catenin、β-catenin在HSC中的表达是否受到cmyb突变的影响,我们利用Tg(cd4Ⅰ:eGFP)转基因胚胎中GFP+来标记HSC,通过QRT-PCR比较了 Cd41-GFP信号分选出的cmybh-3突变体与其同胞的HSC中a-catenin(ctnnaⅠ)、β-catenin(ctnnb1)的表达情况,发现 ctnna1、ctnnb1在cmybhk=3突变体中的表达比在其同胞中表达高;其次,运用morpholino基因敲低拯救实验,分别显微注射ctnna1-MO、ctnnb1-MO 到 Tg(cd41:eGFP)/cmybhk=3intercross的后代中,通过confocal实时观察比较cmybhk=3突变体及其同胞VDA区域的HSC数目以及单位时间内(4h)从VDA区迁出的HSC数目。最后,利用β-catenin的抑制剂氯喹(CQ)处理斑马鱼,观察cmybhk=3突变体VDA区的HSC数目。3.结果通过QRT-PCR实验,发现ctnna1、ctnnb1在cmybhk=-3突变体中的表达比在其同胞中表达高,说明a-catenin、β-catenin可能是cmyb基因的下游调控基因,参与cMyb调控HSC的迁移。显微注射ctnna1-MO和ctnmb1-MO,cmybhk=3纯合突变体VDA区HSC的数目减少到与其同胞相当水平,而且单位时间内迁出的HSC数目也增加。CQ处理后,能促进cmybh=3突变体VDA中滞留的HSC发生迁移,使VDA区域的HSC减少。全文结论第一部分1)CRISPR-Cas9技术介导的斑马鱼基因组定点突变,对1号染色体上的ZKO编号为863～873的11个基因进行基因敲除,获得稳定遗传的F2代突变体。2)在斑马鱼早期造血发育过程中,主要是gfi1aa基因而不是gfi1b基因调控红系的发育过程;3)gfi1aa基因主要通过调控红细胞增殖来影响早期红系造血过程,而gfi1aa基因缺失前体细胞发育正常、红细胞的细胞形态也正常。4)在定向造血阶段,gfi1的家族基因gfi1b会补偿gfi1aa基因的功能。第二部分5)α-catenin、β-catenin是cmyb基因的下游调控基因,共同参与cMyb调控HSC的迁移。6)α-catenin、β-catenin蛋白可以通过改变与细胞骨架的结合,影响胚胎期HSC的迁移。
[Abstract]:Background blood system diseases (such as thalassemia, immunodeficiency and leukemia) have been a serious disease affecting the health of the people in our country. The high incidence of the disease has brought a heavy burden to the health care system of the whole society. The deep reason is that the high incidence of blood diseases reflects the human regulation of hematopoiesis. The molecular and pathological mechanisms of related diseases are poorly understood, because the correct and steady state of the blood system depends on the normal development of various blood cells, and the deficiency of the hematopoietic process leads to a variety of blood cells or vascular dysplasia. Therefore, the development and hematopoiesis process of hematopoietic cells is better understood at the molecular and cellular levels. It will help to gradually clarify the pathogenesis of blood system diseases, thus expanding the application potential of clinical treatment of blood system diseases. In vertebrates, the development process of hematopoietic system is the complex and orderly dynamic process of various blood cell development, mature, and regulated by multiple factors. In spite of the physiological functions of various blood cell components Different, but they have the same ancestor -- hematopoietic stem cells (HSCs). In the development of the hematopoietic system, the early development of hematopoietic stem cells is the core process of the correct production of.HSC, maintenance, proliferation, and smooth migration and localization, the organogenesis of the embryonic stage of the animal body and the stability of the tissue in the adult body. Holding and injury repair are essential for [1,2]. to differentiate from HSC to mature blood cells, experiencing the stage of hematopoietic progenitor cells and the stage of hematopoietic progenitor cells, and eventually differentiating into all lineage mature blood cells. The transcriptional factors play an important role in the development and differentiation of hematopoietic cells. The transcription factors play an important role in the development and differentiation of hematopoietic cells. The transcription factors are enhanced or weakened at different time and space of the cell development, making the hematopoietic stem cells or progenitor cells to a certain extent. Genealogical differentiation.Gfi1 is a transcriptional inhibitor that participates in the regulation of hematopoiesis. The deletion or mutation of the gene is characterized by granulocyte maturation defects and retarding in the early promyelocytic stage. Although leukaemia is not occurring in the Gfi1 knockout mice experiment, the absence of GFI1 function contributes to the occurrence of leukaemia, especially myeloid cells. The occurrence of leukemia, such as AML. classic model organisms such as nematodes and Drosophila, is quite different from human homology, while the traditional model animal mice are very slow and relatively large, and it is difficult to carry out the screening of high throughput hemopoiesis defect mutants. Moreover, mammalian animals are intrauterine, which is not conducive to the observation of embryo hematopoiesis. Zebrafish, as a vertebrate, is highly similar to human beings in organ development, hematopoiesis, and disease. In particular, the early development of the blood and cardiovascular system of zebrafish is very similar to that of human beings, whether the composition of the blood components and the development process are very similar, and the developmental defect mutants of the system still remain. Although it can survive several days, it provides a very favorable condition for people to study the regulation of early embryo hematopoiesis. [3]. in the first part of this paper, TALEN target knockout technique, CRISPR-Cas9 knockout technique, the gfi1aa gene of zebrafish AB strain and GFI1B gene are knocked out successfully, and a variety of different genotypes of gfi1aako mutants are obtained, gfi1bko process The temporal and spatial expression of various genealogical markers in the hematopoiesis process (primary hematopoiesis and permanent hematopoiesis) and the development of various genealogical cells are studied in detail. The purpose of this study is to explore the mechanism of gfi1aa gene and GFI1B gene regulation of erythropoiesis development. The latter part of the paper mainly explores a-catenin, beta -catenin to the embryo period The role of hematopoietic stem cell migration, in order to further clarify the related cell and molecular regulation mechanism of hematopoietic stem cell migration, is divided into two parts: the first part participates in the national "total gene knockout of zebrafish chromosome 1" and the mechanism of gfi1aa regulation of zebrafish blood development; the second part of a-catenin, beta -cat Study on the effect of enin on the migration of hematopoietic stem cells at embryonic stage. Part 1 participates in the national "total gene knockout of zebrafish chromosome 1" and the study on the mechanism of Gfi1 gene regulation of zebrafish hematopoiesis. The purpose of this part is to introduce the CRISPR-Cas9 and TALEN gene editing techniques first, and then use these two techniques with zebrafish as model organisms. The following two tasks were completed: 1) using CRISPR-Cas9 technology to participate in the national "total gene knockout" of zebrafish chromosome 1, and to knock on the 1 chromosome with 11 genes (vps37c, tmem132a, DnaJA1, smu1b, RPS6, btr01, c1h20orf27, muc13a, si:ch211-239f4.6, mucms1). The hereditary F2 generation mutant.2) using the TALEN targeting gene knockout technique, using zebrafish model organisms to knock out the hematopoietic phylogenetic gene gfi1aa, and obtain the gfi1aa mutant of the hematopoietic development defect. At the same time, the CRISPR-Cas9 knockout technique is used to knock out another GFI1B gene of the Gfi1 family and obtain the gfi1bko mutant. Finally, the CRISPR-Cas9 knockout technique is used. Using these two mutants to study the phenotype and function, explore the Gfi1 gene regulating the hematopoietic development cells and the molecular mechanism.2. method 1) mainly using the CRISPR-Cas9 genome editing system, using gRNA target identification and Cas9 nucleic acid cutting. NCBI, Ensemb1 and Smart sites search for the basic information of the related genes, to ZKO compiling. The 11 genes of 863~873 were designed to target the target, and the corresponding primers were synthesized. The corresponding gRNA and Cas9 mRNA were transcribed in vitro, and the mixed microinjection of the wild type AB zebrafish embryos with one-cell was obtained. The zebrafish of the F0 chimera was obtained, and the big FO (Founder) and the wild type AB adult fish were obtained F1 embryos, through the F1 generation embryos. PCR, enzyme digestion and sequencing were used to screen out F0 adult fish with mutation of target loci in germ cells. The F1 of such F0 diplomacy was raised, and the stable genetic zebrafish individual.2 was screened by the sequence of F1 target loci to be screened by scissors, and the TALEN and CRISPR-Cas9 gene knockout techniques were applied to the hematopoiesis development of zebrafish AB strains. The related gene gfi1aa gene and GFI1B gene are knocked out in order to obtain the gfi1aako mutants and gfi1bko mutants. Then the gfi1aa gene and the GFI1B gene regulate the development of the hematopoiesis according to the temporal and spatial expression of various genealogical markers in the hematopoietic and permanent hematopoiesis and the development of each lineage cell. Mechanism.3. result 1) Cas9 gene knockout technique was used to get F1 generation zebrafish with single genotypic mutation, and then F2 generation with AB diplomacy. The zebrafish of F2 generation heterozygous mutant was obtained by PCR, enzyme digestion and sequencing, and a part of the zebrafish of the national zebrafish was given in part, and intercros of the remaining F2 generation mutant zebrafish was then carried out. S, through in situ hybridization, Sultan black B staining, neutral red staining and other methods for phenotypic screening.2), TALEN targeting gene knockout technique was used to knock out zebrafish gfi1aa, and a gfi1aako mutant with 23 bases (5'-TCTTCTCAACAGTCCCCGCTGAG-3') missing was obtained. Phenotypic analysis showed that gfi1aa affected the red fine in the early development of zebrafish hemopoiesis. The development of cell and neutrophils does not affect the development of macrophages. The cytological mechanism that gfi1aa affects the early red lineage is mainly by inhibiting the proliferation of red blood cells, but does not affect the cell morphology of the erythrocyte precursor cells and red cells. In the 50 HPF period, the expression of gfi1aako homozygous hemoglobin was restored to the wild type phase. When.Gfi1b is a homologous gene of gfi1aa, it is also highly expressed in red and megakaryocyte cells. In order to verify that the hematopoietic stage of erythrocyte development is regulated by GFI1B, we use CRISPR-Cas9 knockout technique to obtain the mutant zebra fish of gfi1bko. The results of the double mutant body were found to be in the directional hematopoiesis stage, Gfi1. The family gene GFI1B compensates the function of the gfi1aa gene. Second part a-cateninn, the effect of beta -catenin on the migration of hematopoietic stem cells in embryo stage; 1. the development defects of hematopoietic stem cells (HSC) may cause various blood cell or vascular dysplasia and are closely related to many blood system diseases. The migration of embryonic stem cells is HS C is a necessary process for correct development. However, little is known about the mechanism of HSC migration during embryonic development. There are few molecular studies related to the regulation of embryonic hematopoietic stem cell migration. They are adhesion molecules, cytokines and chemokines [4]., while beta -catenin is a functional protein of the cytoskeleton, and can be used with a-catenin and gamma. Catenin combined with adhesion molecule VE-cadherin, as protein complex involved in intercellular adhesion [5].Takahashi, and other studies found that malignant melanoma B16 cells silenced the beta -catenin gene to promote tumor cells to migrate [6]. and we reported [7] on the regulation of HSC migration in the embryonic stage of cMyb in the early stage, which is unprecedented in the study of cmyb gene function. In this part, we use the cmybhk=3 zebrafish defect mutants as a model to study the mechanism of a-catenin, beta -catenin for the migration and localization of HSC in the embryonic period, and gradually improve the molecular signaling network.2. method for the regulation of HSC development at the embryonic stage, and the detection of a-catenin in zebrafish, beta -catenin in HSC. Whether the expression is affected by cmyb mutation, we use GFP+ to label HSC in Tg (CD4 I: eGFP) transgenic embryos, and compare the expression of cmybh-3 mutant of Cd41-GFP signal and a-catenin (ctnna I) and beta of HSC in Cd41-GFP signal by QRT-PCR. Second, using the morpholino gene knockout experiment, microinjection of ctnna1-MO, ctnnb1-MO to Tg (cd41:eGFP) /cmybhk=3intercross, the number of cmybhk=3 mutants and the number of HSC in the VDA region of the cmybhk=3 and the unit time (4h) from the VDA region were observed in real time by confocal. The -catenin inhibitor chloroquine (CQ) was used to treat zebrafish, and the number of HSC in the cmybhk=3 mutant VDA region was observed by QRT-PCR experiment. The expression of ctnna1 and CTNNB1 in cmybhk=-3 mutants showed higher expression than in their siblings, indicating that a-catenin, beta -catenin may be the downstream regulation gene of the gene. Microinjection of ctnna1-MO and ctnmb1-MO, the number of HSC in the VDA region of the cmybhk=3 homozygous mutant decreased to a considerable level with its compatriots, and the number of HSC in the unit time increased by.CQ processing, which could promote the migration of HSC in the cmybh=3 mutant VDA, and reduce the HSC in VDA region. The first part of the full text conclusion 1) Directed mutagenesis of zebrafish genome, gene knockout of 11 genes with ZKO number of 863~873 on chromosome 1, and a stable genetic F2 generation mutant.2). During the early hematopoiesis of zebrafish, the major gfi1aa gene was not the GFI1B gene regulating the development of the red system; 3) the gfi1aa gene was mainly controlled by the regulation of red fine. The cell proliferation affects the early erythropoiesis process, while the gfi1aa gene deletion precursor cells develop normally, the cell morphology of the red cells is also normal.4). The family gene GFI1B of Gfi1 will compensate for the function of the gfi1aa gene. Second part 5) alpha -catenin, the beta -catenin is the downstream regulation gene of the cmyb gene, and participates in cMyb regulation HSC. The migration of.6) alpha -catenin and beta -catenin can affect the migration of HSC in embryonic stage by changing the binding with cytoskeleton.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R331

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