人胚胎干细胞向造血细胞诱导分化的研究
发布时间：2018-07-13 14:45
【摘要】： 血细胞输注和造血干细胞(hematopoietic stem cells ,HSCs)移植是细胞治疗的常用手段,可用来治疗恶性血液疾病、感染性疾病、遗传性疾病、重症免疫缺陷、AIDS等多种疾病。然而目前血细胞来源紧张及污染问题给其临床使用的安全性和广泛性带来了极大挑战。因此人们期望更为安全、有效和经济的血细胞资源。随着干细胞研究及其相关领域的生物学技术的快速发展,以人胚胎干细胞(human embryonic stem cells,hESCs)为启动细胞的造血干细胞工程为临床输血和干细胞的移植带来了新希望。 目前,研究者主要使用造血发育相关因子或造血微环境的基质细胞促进人胚胎干细胞向造血细胞分化。虽然上述诱导方法在诱导胚胎干细胞向造血细胞分化上取得了良好的效果,但仍然存在着一些难以克服的局限性:鼠源饲养层是目前常用的较为高效的诱导方法;然而鼠源饲养层所带来的异源污染极大地限制了该研究的临床应用前景。诱导中使用的各种细胞因子多为基因工程产品,价格昂贵,可在研究中使用却不能大量推广。因此我们希望建立一种新的诱导方案既可以高效诱导人胚胎干细胞向造血细胞分化,又可以避免异源污染,同时可降低实验成本。 造血发生经历3个阶段:卵黄囊造血、胎肝造血和骨髓造血。人卵黄囊造血时期为4～6周;胎肝造血时期为6～22周;骨髓造血从22周至出生。在胚胎发育的不同阶段,不同的造血微环境对造血发生发挥着重要作用。在利用ESCs体外定向分化为造血细胞的模型中,诱导条件(微环境)的选择策略是研究造血发生和分化调控机制的重要环节。胎肝是造血发育的主要位点,该组织的微环境为造血细胞的生成提供了必要的条件。理论上来说胎肝造血微环境对胚胎干细胞向造血细胞的分化应该会有很好的诱导支持作用。因此在本实验中我们模拟胎肝造血微环境,联合使用15周人胎肝基质细胞和人胎肝组织提取物诱导人胚胎干细胞分化为造血细胞,并比较该方法与人胎肝基质细胞诱导法及人胎肝基质细胞/细胞因子诱导法之间的差别,旨在建立一种高效安全的造血细胞诱导体系。 首先,我们分离15周人流产胎儿胎肝基质细胞并制备胎肝组织细胞提取物,使用半定量RT-PCR和流式细胞技术分析了胎肝基质细胞表面标志及基因表达情况。结果表明人胎肝基质细胞表达间质细胞表面标志CD90、CD29,不表达造血细胞表面标志CD34和CD45;此外,人胎肝基质细胞表达造血发育支持因子EPO、Flt-3和SCF,但随着传代时间的延长这些因子的表达水平逐渐降低。由此我们选择前3代的胎肝基质细胞作为胚胎干细胞造血诱导的饲养层。 之后,我们将人胚胎干细胞培养于低黏附性培养皿中诱导形成拟胚体(human embryoid bodies ,hEBs),并用骨形成蛋白4(Bone morphogenetic proteins 4, BMP4)对拟胚体进行了处理,通过检测拟胚体向中胚层发育的情况,选择了适当培养天数的拟胚体,采用三种不同的诱导方法进行诱导分化。在诱导的过程中,我们收集不同培养天数的细胞,应用流式细胞技术检测了分化体系中CD34、CD45等造血表面抗原的表达情况,以确定各组的造血分化情况。结果表明三种不同的诱导方法均能促进人胚胎干细胞向造血细胞定向分化。但在不添加细胞因子和细胞提取物的情况下,胎肝基质细胞诱导人胚胎干细胞向造血细胞分化的能力较弱,在诱导的10天中CD34+细胞比率与CD45+细胞比率均低于10%。在添加细胞因子之后,人胚胎干细胞向造血细胞分化的能力明显增强,其中CD34+细胞最高可达24.68%, CD45+细胞最高可达13.57%。联合使用胎肝基质细胞与胎肝细胞提取物则能更为高效的诱导人胚胎干向造血细胞发育,其中CD34+细胞最高可达32.73%, CD45+细胞最高可达27.96%。此外,我们收集诱导10天的细胞,应用RT-PCR检测了细胞造血相关基因的表达变化情况;应用克隆形成实验检测了细胞的造血克隆形成能力;应用Wright—Giemsa染色观测了细胞的形态;应用免疫荧光染色技术检测了诱导集落造血表面抗原的表达情况。结果发现,诱导产生的细胞具有造血细胞的一般特性,表达造血相关基因AML、SCL、GATA-1;在半固体培养基中可分化为不同种类的造血克隆。但不同诱导体系中产生的细胞造血基因的表达量与造血克隆的生成能力不同,胎肝基质细胞/胎肝细胞提取物体系产生细胞AML、SCL、GATA-1的表达量及形成的造血克隆最多,且以红系克隆为主。在上述研究的基础上,我们进一步探讨造血祖细胞向红系发育的机制,并建立了高效的造血祖细胞向红细胞的分化体系,为以胚胎干细胞为启动细胞诱导分化规模化的产生红细胞奠定一定的基础。红细胞的产生受到造血因子和造血干细胞自身内在基因的共同调控,其中促红细胞生成素(erythropoietin,EPO)是其产生的最重要因子。细胞因子信号转导抑制因子-3(suppressor of cytokine signaling-3 ,SOCS-3)最早发现于1997年,有研究证明EPO受体上有SOCS-3的高亲和结合位点,SOCS-3对EPO具有负调控作用。因此,我们推测降低胚胎干细胞或造血祖细胞中SOCS-3基因的表达水平有利于它们向红系的发育。在本实验中我们选择具有造血干细胞特性的人红白血病细胞株K562作为研究对象,构建了SOCS-3慢病毒siRNA干涉载体,转染K562细胞。根据绿色荧光蛋白的表达进行流式分选后,我们获得了高表达慢病毒干涉载体的细胞。实时荧光定量PCR和Western-blot检测了转染细胞中SOCS-3基因的干涉效率,结果显示与对照组相比,siRNA干涉后K562细胞SOCS-3基因的表达量仅为其相对表达量的22.1%,干涉效率77.9%;Western -blot结果显示SOCS-3在蛋白质水平表达也明显受抑制。我们进一步对SOCS-3基因沉默后的K562细胞进行了诱导分化,并采用联苯胺染色法检测K562细胞向红系分化比例变化,免疫荧光染色检测细胞表面抗原的变化,RT-PCR检测造血相关基因的变化,发现SOCS-3沉默后K562细胞向红系的发育能力显著提高。 综上所述,在本研究中我们采用三种不同方法诱导人胚胎干细胞分化为造血细胞,并证明联合使用胎肝基质细胞和胎肝组织细胞提取物是较好的诱导方法,该方法可高效诱导胚胎干细胞向造血细胞分化,且避免了鼠源饲养层所带来的异源污染,降低了实验成本。在此基础上我们通过构建慢病毒干涉载体的方法,稳定干涉了K562细胞中SOCS-3表达,建立了高效的造血祖细胞向红系细胞诱导体系,证明了SOCS-3基因沉默有利于造血祖细胞向红系的发育。上述研究为深入探讨造血细胞发育调控机制以及以胚胎干细胞或造血干细胞为启动细胞大规模的诱导产生红细胞体系的建立奠定了基础。
[Abstract]:Blood cell infusion and hematopoietic stem cells (HSCs) transplantation are the common means of cell therapy, which can be used to treat many diseases, such as malignant blood diseases, infectious diseases, hereditary diseases, severe immunodeficiency, AIDS and so on. However, the problem of blood cell source tension and pollution to the clinical use is safe and extensive. It brings great challenges. So people expect more safe, effective and economical blood cells. With the rapid development of stem cell research and its related fields, human embryonic stem cells (human embryonic stem cells, hESCs) for the hematopoietic stem cell engineering that start the cells for clinical transfusions and stem cell transplantation New hope.
At present, researchers mainly use hematopoietic growth related factors or stromal cells in hematopoietic microenvironment to promote human embryonic stem cells to differentiate into hematopoietic cells. Although the afore-mentioned induction methods have achieved good results in inducing embryonic stem cells to differentiate into hematopoietic cells, there are still some insuperable limitations: the rat feeder layer is the eye. However, the heterologous pollution caused by the rat feeder layer greatly restricts the prospect of the clinical application. The various cytokines used in the induction are mostly genetic engineering products, which are expensive and can not be widely used in the study. Therefore, we hope to establish a new induction scheme. It can effectively induce the differentiation of human embryonic stem cells into hematopoietic cells and avoid heterogenous pollution, and at the same time, it can reduce the cost of experiments.
Hematopoiesis occurs in 3 stages: yolk sac hematopoiesis, fetal liver hematopoiesis and marrow hematopoiesis. Human yolk sac hematopoiesis period is 4~6 weeks; fetal liver hematopoiesis period is 6~22 weeks; bone marrow hematopoiesis is from 22 weeks to birth. In different stages of embryo development, different hematopoietic microenvironment plays an important role in hematopoiesis. The differentiation of hematopoiesis in vitro by ESCs is made in vitro. In the blood cell model, the selection strategy of the induction condition (microenvironment) is an important link in the study of the regulation mechanism of hematopoiesis and differentiation. Fetal liver is the main locus of hematopoiesis, and the microenvironment of this tissue provides the necessary conditions for the generation of hematopoietic cells. In this experiment, we simulated fetal liver hematopoietic microenvironment, combined with 15 weeks human fetal liver stromal cells and human fetal liver tissues to induce human embryonic stem cells to differentiate into hematopoietic cells, and compare this method with human fetal liver stromal cell induction and human fetal liver stromal cells / cell causes. The difference between sub induction methods is to establish an efficient and safe induction system for hematopoietic cells.
First, we isolated fetal liver stromal cells and prepared fetal liver tissue extracts for 15 weeks. The surface markers and gene expressions of fetal liver stromal cells were analyzed by semi quantitative RT-PCR and flow cytometry. The results showed that human fetal liver stromal cells expressed CD90, CD29, and no expression of hematopoietic cell surface. In addition, human fetal liver stromal cells express hematopoietic growth support factor EPO, Flt-3 and SCF, but the expression level of these factors gradually decreases with the prolongation of passage time. Therefore, we choose fetal liver stromal cells in the first 3 generations as the feeding layer induced by embryonic stem cell hematopoiesis.
After that, we induce the human embryonic stem cells to form the human embryoid bodies (hEBs) in the low adhesion culture dish, and use the bone forming protein 4 (Bone morphogenetic proteins 4, BMP4) to deal with the embryoid body. By detecting the development of the mesoembryonic body to the middle embryo layer, the proper culture number of the pseudo embryoid body is selected. Three different induction methods were used to induce differentiation. In the process of induction, we collected the cells with different days of culture and detected the expression of CD34, CD45 and other hematopoietic surface antigens in the differentiation system by flow cytometry to determine the hematopoietic differentiation in each group. The results showed that three different induction methods could promote the human embryo. The fetal stem cells differentiate into hematopoietic cells, but without the addition of cytokines and cell extracts, the ability of fetal liver stromal cells to induce human embryonic stem cells to differentiate into hematopoietic cells is weak. The ratio of CD34+ cells to CD45+ cells in the 10 days of induction is lower than that of 10%. in the addition of cytokines. The ability of the differentiation of blood cells increased obviously, of which the highest CD34+ cells could reach 24.68%, the highest of CD45+ cells could reach 13.57%. and the combination of fetal liver stromal cells and fetal hepatocyte extract could induce the development of human embryonic stem to hematopoietic cells more efficiently, of which the maximum of CD34+ cells was up to 32.73%, and the maximum of CD45+ cells could reach 27.96%.. The cells which were induced for 10 days were collected and the expression of hematopoiesis related genes was detected by RT-PCR. The ability of the hematopoietic clone formation was detected by the cloning and formation test. The morphology of the cells was observed by Wright Giemsa staining, and the expression of the induced colony hematopoietic surface antigen was detected by immunofluorescence staining. The results showed that the induced cells had general characteristics of hematopoietic cells, expressed hematopoietic related genes AML, SCL, GATA-1, and differentiated into different kinds of hematopoietic clones in the semisolid medium, but the expression of hematopoietic genes produced in different induction systems was different from that of hematopoietic clones, and the fetal liver stromal cells / fetal liver were fine. The cell extract system produces AML, SCL, GATA-1 and the most hematopoietic clones, which are mainly red clones. On the basis of the above study, we further explore the mechanism of hematopoietic progenitor cells to the erythroid development, and establish a highly efficient hematopoietic progenitor cell differentiation system for the initiation of embryonic stem cells. The production of erythrocytes is controlled by the co regulation of hematopoietic and hematopoietic stem cells's own intrinsic genes, in which erythropoietin (erythropoietin, EPO) is the most important factor in its production. Cytokine signal transduction inhibitor -3 (suppressor of cytokine signali) NG-3, SOCS-3) was first discovered in 1997. Studies have shown that the EPO receptor has a high affinity binding site for SOCS-3, and SOCS-3 has a negative regulatory effect on EPO. Therefore, we speculate that the reduction of the expression level of the SOCS-3 gene in embryonic stem cells or hematopoietic progenitor cells is beneficial to the development of their erythroid system. In this experiment, we chose to have hematopoietic stem cells. The cell characteristic human erythroleukemia cell line K562 was used as the research object to construct the SOCS-3 lentivirus siRNA interference carrier and transfect the K562 cells. We obtained the cells with high expression of the lentivirus interference carrier based on the expression of the green fluorescent protein. The real-time fluorescence quantitative PCR and Western-blot were used to detect the SOCS-3 base in the transfected cells. The results showed that the expression of SOCS-3 gene in K562 cells after siRNA interference was only 22.1% of the relative expression and 77.9% in the interference efficiency compared with the control group, and the Western -blot results showed that the expression of SOCS-3 at the protein level was also obviously inhibited. We further induced the differentiation of K562 cells after the SOCS-3 gene silencing, The differentiation ratio of K562 cells to erythroid differentiation was detected by diphenyl amine staining, and the changes of cell surface antigen were detected by immunofluorescence staining. The changes of hematopoiesis related genes were detected by RT-PCR. It was found that the development ability of K562 cells to the red system was significantly improved after SOCS-3 silencing.
In summary, in this study, we used three different methods to induce human embryonic stem cells to differentiate into hematopoietic cells, and proved that combined use of fetal liver stromal cells and fetal liver tissue cells extracts is a good induction method. This method can effectively induce embryonic stem cells to differentiate into hematopoietic cells and avoid the result of rat feeder layer. Heterogenous pollution reduces the cost of experiment. On this basis, we establish a highly efficient hematopoietic progenitor cell induction system by interfering with the expression of SOCS-3 in K562 cells by constructing the lentivirus interferometric vector. It is proved that the silence of SOCS-3 gene is beneficial to the development of hematopoietic progenitor cells. The mechanism of the regulation of hematopoietic cell development and the establishment of a large-scale induction of erythrocyte system by embryonic stem cells or hematopoietic stem cells is the basis for the establishment of a red cell system.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329
[Abstract]:Blood cell infusion and hematopoietic stem cells (HSCs) transplantation are the common means of cell therapy, which can be used to treat many diseases, such as malignant blood diseases, infectious diseases, hereditary diseases, severe immunodeficiency, AIDS and so on. However, the problem of blood cell source tension and pollution to the clinical use is safe and extensive. It brings great challenges. So people expect more safe, effective and economical blood cells. With the rapid development of stem cell research and its related fields, human embryonic stem cells (human embryonic stem cells, hESCs) for the hematopoietic stem cell engineering that start the cells for clinical transfusions and stem cell transplantation New hope.
At present, researchers mainly use hematopoietic growth related factors or stromal cells in hematopoietic microenvironment to promote human embryonic stem cells to differentiate into hematopoietic cells. Although the afore-mentioned induction methods have achieved good results in inducing embryonic stem cells to differentiate into hematopoietic cells, there are still some insuperable limitations: the rat feeder layer is the eye. However, the heterologous pollution caused by the rat feeder layer greatly restricts the prospect of the clinical application. The various cytokines used in the induction are mostly genetic engineering products, which are expensive and can not be widely used in the study. Therefore, we hope to establish a new induction scheme. It can effectively induce the differentiation of human embryonic stem cells into hematopoietic cells and avoid heterogenous pollution, and at the same time, it can reduce the cost of experiments.
Hematopoiesis occurs in 3 stages: yolk sac hematopoiesis, fetal liver hematopoiesis and marrow hematopoiesis. Human yolk sac hematopoiesis period is 4~6 weeks; fetal liver hematopoiesis period is 6~22 weeks; bone marrow hematopoiesis is from 22 weeks to birth. In different stages of embryo development, different hematopoietic microenvironment plays an important role in hematopoiesis. The differentiation of hematopoiesis in vitro by ESCs is made in vitro. In the blood cell model, the selection strategy of the induction condition (microenvironment) is an important link in the study of the regulation mechanism of hematopoiesis and differentiation. Fetal liver is the main locus of hematopoiesis, and the microenvironment of this tissue provides the necessary conditions for the generation of hematopoietic cells. In this experiment, we simulated fetal liver hematopoietic microenvironment, combined with 15 weeks human fetal liver stromal cells and human fetal liver tissues to induce human embryonic stem cells to differentiate into hematopoietic cells, and compare this method with human fetal liver stromal cell induction and human fetal liver stromal cells / cell causes. The difference between sub induction methods is to establish an efficient and safe induction system for hematopoietic cells.
First, we isolated fetal liver stromal cells and prepared fetal liver tissue extracts for 15 weeks. The surface markers and gene expressions of fetal liver stromal cells were analyzed by semi quantitative RT-PCR and flow cytometry. The results showed that human fetal liver stromal cells expressed CD90, CD29, and no expression of hematopoietic cell surface. In addition, human fetal liver stromal cells express hematopoietic growth support factor EPO, Flt-3 and SCF, but the expression level of these factors gradually decreases with the prolongation of passage time. Therefore, we choose fetal liver stromal cells in the first 3 generations as the feeding layer induced by embryonic stem cell hematopoiesis.
After that, we induce the human embryonic stem cells to form the human embryoid bodies (hEBs) in the low adhesion culture dish, and use the bone forming protein 4 (Bone morphogenetic proteins 4, BMP4) to deal with the embryoid body. By detecting the development of the mesoembryonic body to the middle embryo layer, the proper culture number of the pseudo embryoid body is selected. Three different induction methods were used to induce differentiation. In the process of induction, we collected the cells with different days of culture and detected the expression of CD34, CD45 and other hematopoietic surface antigens in the differentiation system by flow cytometry to determine the hematopoietic differentiation in each group. The results showed that three different induction methods could promote the human embryo. The fetal stem cells differentiate into hematopoietic cells, but without the addition of cytokines and cell extracts, the ability of fetal liver stromal cells to induce human embryonic stem cells to differentiate into hematopoietic cells is weak. The ratio of CD34+ cells to CD45+ cells in the 10 days of induction is lower than that of 10%. in the addition of cytokines. The ability of the differentiation of blood cells increased obviously, of which the highest CD34+ cells could reach 24.68%, the highest of CD45+ cells could reach 13.57%. and the combination of fetal liver stromal cells and fetal hepatocyte extract could induce the development of human embryonic stem to hematopoietic cells more efficiently, of which the maximum of CD34+ cells was up to 32.73%, and the maximum of CD45+ cells could reach 27.96%.. The cells which were induced for 10 days were collected and the expression of hematopoiesis related genes was detected by RT-PCR. The ability of the hematopoietic clone formation was detected by the cloning and formation test. The morphology of the cells was observed by Wright Giemsa staining, and the expression of the induced colony hematopoietic surface antigen was detected by immunofluorescence staining. The results showed that the induced cells had general characteristics of hematopoietic cells, expressed hematopoietic related genes AML, SCL, GATA-1, and differentiated into different kinds of hematopoietic clones in the semisolid medium, but the expression of hematopoietic genes produced in different induction systems was different from that of hematopoietic clones, and the fetal liver stromal cells / fetal liver were fine. The cell extract system produces AML, SCL, GATA-1 and the most hematopoietic clones, which are mainly red clones. On the basis of the above study, we further explore the mechanism of hematopoietic progenitor cells to the erythroid development, and establish a highly efficient hematopoietic progenitor cell differentiation system for the initiation of embryonic stem cells. The production of erythrocytes is controlled by the co regulation of hematopoietic and hematopoietic stem cells's own intrinsic genes, in which erythropoietin (erythropoietin, EPO) is the most important factor in its production. Cytokine signal transduction inhibitor -3 (suppressor of cytokine signali) NG-3, SOCS-3) was first discovered in 1997. Studies have shown that the EPO receptor has a high affinity binding site for SOCS-3, and SOCS-3 has a negative regulatory effect on EPO. Therefore, we speculate that the reduction of the expression level of the SOCS-3 gene in embryonic stem cells or hematopoietic progenitor cells is beneficial to the development of their erythroid system. In this experiment, we chose to have hematopoietic stem cells. The cell characteristic human erythroleukemia cell line K562 was used as the research object to construct the SOCS-3 lentivirus siRNA interference carrier and transfect the K562 cells. We obtained the cells with high expression of the lentivirus interference carrier based on the expression of the green fluorescent protein. The real-time fluorescence quantitative PCR and Western-blot were used to detect the SOCS-3 base in the transfected cells. The results showed that the expression of SOCS-3 gene in K562 cells after siRNA interference was only 22.1% of the relative expression and 77.9% in the interference efficiency compared with the control group, and the Western -blot results showed that the expression of SOCS-3 at the protein level was also obviously inhibited. We further induced the differentiation of K562 cells after the SOCS-3 gene silencing, The differentiation ratio of K562 cells to erythroid differentiation was detected by diphenyl amine staining, and the changes of cell surface antigen were detected by immunofluorescence staining. The changes of hematopoiesis related genes were detected by RT-PCR. It was found that the development ability of K562 cells to the red system was significantly improved after SOCS-3 silencing.
In summary, in this study, we used three different methods to induce human embryonic stem cells to differentiate into hematopoietic cells, and proved that combined use of fetal liver stromal cells and fetal liver tissue cells extracts is a good induction method. This method can effectively induce embryonic stem cells to differentiate into hematopoietic cells and avoid the result of rat feeder layer. Heterogenous pollution reduces the cost of experiment. On this basis, we establish a highly efficient hematopoietic progenitor cell induction system by interfering with the expression of SOCS-3 in K562 cells by constructing the lentivirus interferometric vector. It is proved that the silence of SOCS-3 gene is beneficial to the development of hematopoietic progenitor cells. The mechanism of the regulation of hematopoietic cell development and the establishment of a large-scale induction of erythrocyte system by embryonic stem cells or hematopoietic stem cells is the basis for the establishment of a red cell system.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329
【参考文献】
相关期刊论文 前3条
1 向国春,张佳思,成晓玲,黎儒清,赵树铭;输注去白细胞红细胞悬液预防非溶血性发热性输血反应的临床应用分析[J];第三军医大学学报;2001年07期
2 张祖s，
本文编号：2119748
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2119748.html
最近更新
教材专著
