胚胎干细胞或胚胎样干细胞向血管细胞的分化及调控机制研究

发布时间：2018-01-02 08:00

本文关键词：胚胎干细胞或胚胎样干细胞向血管细胞的分化及调控机制研究　出处：《中南大学》2010年博士论文　论文类型：学位论文

【摘要】： 血管系统是人类最大,分布最广的系统。血管细胞主要包括内皮细胞和平滑肌细胞。血管细胞的功能异常可以导致动脉粥样硬化,心肌梗塞,脑中风等血管疾病。胚胎干细胞来源于囊胚内细胞团,是具有无限增殖能力和向各胚层细胞分化能力的全能干细胞。胚胎干细胞向内皮细胞或平滑肌细胞分化的研究对血管细胞的发育生物学,心血管药物的筛选,并可望为细胞移植治疗提供细胞来源。本研究论文主要进行小鼠和人类的胚胎干细胞(ESCs)及胚胎干细胞样细胞(iPS细胞)向内皮细胞(ECs)和平滑肌细胞(SMCs)分化的体系构建及相关机制研究,包括以下五个方面：1、小鼠骨髓内皮细胞条件培养液对小鼠胚胎干细胞向内皮细胞分化的诱导作用；2、人类胚胎干细胞来源的内皮样细胞与脐血来源的内皮祖细胞和脐静脉内皮细胞的比较；3、iPS细胞向内皮细胞及平滑肌细胞的分化；4、平滑肌细胞分化过程中促进分化的转录因子的筛选及调控机制的初步研究；5、锌指蛋白297B协同Myocardin调节平滑肌细胞的分化。第一章小鼠骨髓内皮细胞条件培养液促进鼠胚胎干细胞向内皮细胞的诱导分化目的：小鼠骨髓内皮细胞的条件培养液在以前的实验中被证明能促进造血细胞的增殖及分化,促进骨髓内皮细胞的生长。本实验研究内皮细胞条件培养基(mEC-CM)对小鼠胚胎干细胞向内皮细胞分化的促进作用及分化的内皮细胞的纯化。方法：在这个研究中,我们用mEC-CM诱导mESCs向内皮细胞前体细胞Flkl+细胞分化,并将诱导分化的效率同细胞因子(VEGF,EGF,bFGF和IGF-1)诱导的效率相比较。我们还从分化的mESCs中机械挑选了吞噬DiI-Ac-LDL的鹅卵石样细胞,分析其生物学特性。结果：mEC-CM能明显促进mESCs向Flkl+细胞的分化,其效率与细胞因子的诱导相似,mEC-CM与细胞因子组合没有协同诱导作用。mEC-CM的诱导结合机械挑选DiI-Ac-LDL标记阳性的鹅卵石样细胞的方法,能纯化分化的内皮样细胞,表达内皮细胞标志,结合UEA1,并能在体外形成血管样结构。结论：mEC-CM能诱导mESCs向内皮细胞的分化。机械挑选DiI-Ac-LDL标记阳性的鹅卵石样细胞的方法能纯化mESCs分化的内皮样细胞。第二章胚胎干细胞来源的内皮细胞的诱导分化及其与人脐静脉内皮细胞和脐血内皮祖细胞的比较目的：从人类胚胎干细胞分化系统中分选出内皮前体细胞(KDR+细胞),研究其特性,并与脐血来源的内皮祖细胞和脐静脉内皮细胞进行比较。方法：用本研究所建立的人类胚胎干细胞株chESC-1, chESC-3, chESC-8, chESC-20和chESC-22,自发分化成9天的拟胚体,磁珠分选出KDR+细胞,在内皮细胞培养基培养后,检测其内皮细胞标志的表达,生长特性,及其内皮细胞功能,并与脐血内皮祖细胞和脐静脉内皮细胞进行比较。结果：从9天EBs中分选出KDR+细胞在内皮细胞培养基中培养后,与成体内皮细胞相比较,胚胎干细胞来源的KDR+细胞表达更多的内皮祖细胞标志如CD133,CD34,不表达成熟内皮细胞标志vWF,能结合UEA1,吞噬DiI-Ac-LDL,并能在matrigel上形成血管样结构。在体外培养过程中,KDR+细胞的祖细胞标志逐渐减少,出现成熟内皮细胞标志。结论：人胚胎干细胞株能向内皮细胞分化,分选得到的KDR+细胞在内皮细胞培养条件下与成体内皮细胞比较显示其内皮祖细胞特性,并能在体外培养中向成熟内皮细胞分化。第三章iPS细胞向内皮细胞和平滑肌细胞的诱导分化目的：成纤维细胞转染Oct4, c-Myc, Sox2和Klf4四种因子得到的诱导的多能干细胞(induced pluripotent stem cells,iPS)是一种胚胎干细胞样细胞,能无限增殖并具有多向分化潜能,较之胚胎干细胞,iPS细胞避免了伦理问题,且能建立患者特异性的细胞株,具有很高的临床应用价值。本研究探讨小鼠iPS细胞向血管细胞(内皮细胞和平滑肌细胞)分化的能力。方法：采用单层贴壁诱导及流式细胞术进行Flkl+细胞分化与分选,进而与OP9基质细胞共培养,并用VE-cadherin为标志纯化内皮细胞(09-EC)；用高剂量反式视黄酸(RA)诱导iPS向平滑肌细胞分化。检测分化细胞的基因表达,免疫标记和细胞功能,与普通小鼠胚胎干细胞的分化效率相比较。结果：小鼠iPS细胞可以向内皮细胞和平滑肌细胞分化,分化效率与普通小鼠胚胎干细胞相似。分化的内皮细胞能表达CD31,CD144等表面标志,吞噬DiI-Ac-LDL,并能在matrigel上形成血管。分化的平滑肌细胞能表达SMAa, SMMHC等标志,在碳酰胆碱刺激下有收缩功能。分化过程中内皮或平滑肌细胞特异性基因表达上调,而Oct4、Sox2、Klf4和c-Myc这四种诱导iPS细胞形成的转录因子的表达显著下调。结论：小鼠iPS细胞能向内皮细胞和平滑肌细胞诱导分化,其分化潜能与小鼠胚胎干细胞相似。第四章平滑肌细胞重要调节因子Myocardin的协同转录因子的筛选目的：平滑肌细胞的增殖分化与血管内膜增生,冠状动脉狭窄等血管病理密切相关,Myocardin是平滑肌分化过程中不可缺少的一个协同转录因子,本实验希望通过筛选与Myocardin结合的蛋白,找到在平滑肌分化过程中新的重要的转录因子或协同转录因子,研究其在平滑肌分化过程中的功能及所属信号通路,进而更深入了解平滑肌分化的分子机制。方法：用荧光素酶活性实验筛选1170个转录因子中能上调Myocardin表达的转录因子,并用免疫共沉淀等方法进一步确认候选因子与Myocardin蛋白水平的物理结合,通过生物信息学分析等方法筛选出候选因子,在小鼠胚胎干细胞向平滑肌细胞分化模型中确定其在平滑肌细胞分化中的调节作用。结果：我们从1170个转录因子中筛选出ZNF297B,RAI14和MAGED1等转录因子,确定其在平滑肌中高表达,在平滑肌分化过程中上调,并能与Myocardin直接结合,其过表达能促进平滑肌分化基因的表达。结论：ZNF297B,RAI14和MAGED1等转录因子可能是调节平滑肌分化的重要转录因子。第五章锌指蛋白297B协同Myocardin调节平滑肌细胞的分化目的：探讨锌指蛋白297B(ZNF297B)在平滑肌细胞分化过程中的表达及相关机制。方法：用定量实时PCR检测ZNF297B在人,大鼠,小鼠平滑肌细胞中体内／体外的表达量及在平滑肌细胞体内和体外分化或增殖过程中的表达变化；构建ZNF297B-Myc表达质粒,免疫荧光检测ZNF297B-Myc在大鼠平滑肌细胞中的表达分布；用荧光素酶活性检测及免疫共沉淀实验检测ZNF297B与Myocardin的结合及相互作用。结果：ZNF297B在平滑肌细胞中特异性高表达,在小鼠胚胎干细胞向平滑肌细胞分化过程中表达上调,在PDGFBB刺激的平滑肌细胞增殖过程中下调,在大鼠颈动脉球囊损伤内膜修复过程中表达先上调后下降。ZNF297B主要分布于细胞核及核周的细胞质。ZNF297B能与Myocardin蛋白结合,荧光素酶活性检测显示ZNF297B的表达受Myocardin的调控。结论：ZNF297B可能在平滑肌细胞分化过程中起到重要正向调控作用,这一作用很可能是通过ZNF297B与Myocardin的协同作用来完成。
[Abstract]:Vascular system is the largest and most widely distributed system in human beings. Vascular cells mainly consist of endothelial cells and smooth muscle cells. Dysfunction of vascular cells can lead to atherosclerosis, myocardial infarction, stroke and other vascular diseases.
Embryonic stem cells derived from the inner cell mass, has infinite proliferation ability and the ability to differentiate the endoderm cells. Stem cells from embryonic stem cells on endothelial cells or smooth muscle cells on the differentiation of vascular cells in developmental biology, screening of cardiovascular drugs, and is expected to provide a cell source for cell transplantation therapy. This research paper the main of mouse and human embryonic stem cells (ESCs) and embryonic stem cells (iPS cells) into endothelial cells (ECs) and smooth muscle cells (SMCs) on the construction and mechanism of differentiation of the system, including the following five aspects: 1, fluid on mouse embryonic stem cells to induce differentiation of endothelial cells the culture conditions of mouse bone marrow endothelial cells; 2, comparison of human embryonic stem cells derived endothelial like cells and umbilical cord blood derived endothelial progenitor cells and human umbilical vein endothelial cells; iPS cell in 3 The differentiation of skin cells and smooth muscle cells; 4, the screening and regulation mechanism of transcription factors promoting differentiation during smooth muscle cell differentiation; 5, zinc finger protein 297B co regulated Myocardin with smooth muscle cell differentiation.
In Chapter 1, the conditioned medium of mouse bone marrow endothelial cells promotes the induced differentiation of mouse embryonic stem cells to endothelial cells
Objective: mouse bone marrow endothelial cell conditioned medium has been shown to promote the proliferation and differentiation of hematopoietic cells in the previous experiment, bone marrow endothelial cell growth conditions. The experimental study of endothelial cell culture medium (mEC-CM) on mouse embryonic stem cells into endothelial cells and promote the purification effect and differentiation into endothelial cells..
Methods: in this study, we use mEC-CM mESCs to induce endothelial progenitor cells differentiation of Flkl+ cells, and the efficiency of differentiation with cytokines (VEGF, EGF, bFGF and IGF-1) induced efficiency. We also compared the differentiation of mESCs from mechanical selected cobblestone phagocytosis of DiI-Ac-LDL, analysis its biological characteristics.
Results: mEC-CM could significantly promote the differentiation of mESCs into Flkl+ cells, the efficiency of similar induction with cytokines, mEC-CM and cytokine combination method induced no synergistic combination of mechanical DiI-Ac-LDL markers selected cobblestone positive cells induced by.MEC-CM, to purification of endothelial cells the expression of endothelial cell markers, combined with UEA1. And can form vascular like structure in vitro.
Conclusion: mEC-CM can induce mESCs to differentiate into endothelial cells. Mechanical selection of DiI-Ac-LDL labeled positive cobblestone cells can purify mESCs differentiated endothelial like cells.
Induction and differentiation of endothelial cells derived from embryonic stem cells in the second chapter and comparison with human umbilical vein endothelial cells and umbilical cord blood endothelial progenitor cells
Objective: to identify endothelial progenitor cells (KDR+ cells) from human embryonic stem cell differentiation system and study their characteristics, and compare them with umbilical cord blood derived endothelial progenitor cells and umbilical vein endothelial cells.
Methods: using human embryonic stem cells chESC-1, this study established the chESC-3, chESC-8, chESC-20 and chESC-22, the spontaneous differentiation into embryoid bodies 9 days, magnetic beads selected KDR+ cells cultured in endothelial cells, expression of growth characteristics, detection of endothelial cell markers, and the function of endothelial cell, and compared with umbilical cord blood endothelial progenitor cells and human umbilical vein endothelial cells.
Results: from the 9 day EBs were selected KDR+ cells were cultured in endothelial cell medium, compared with adult endothelial cells, more endothelial progenitor cell markers such as CD133, the expression of embryonic stem cell derived KDR+ CD34 cells, expression of mature endothelial cell marker vWF, with UEA1, DiI-Ac-LDL and phagocytosis. The formation of vessel like structures in Matrigel. In the process of in vitro culture, progenitor cell markers of KDR+ cells gradually decreased, while mature endothelial markers.
Conclusion: the human embryonic stem cell line can differentiate into endothelial cells. The KDR+ cells obtained from the differentiated cells show endothelial progenitor cell characteristics compared with adult endothelial cells under endothelial cell culture conditions, and can differentiate into mature endothelial cells in vitro.
Induction and differentiation of iPS cells to endothelial cells and smooth muscle cells in the third chapter
Objective: fibroblast cells transfected with Oct4, c-Myc, Sox2 and Klf4 four kinds of cytokine induced pluripotent stem cells (induced pluripotent stem cells, iPS) is an embryonic stem cell like cells, can have the potential of multi-directional differentiation and proliferation, compared to embryonic stem cells, iPS cells to avoid the ethical problems, and the establishment of patient specific cells, has high clinical value. This study of mouse iPS cells into vascular cells (endothelial cells and smooth muscle cells) differentiation ability.
Methods: the monolayer induced by flow cytometry and Flkl+ cell differentiation and sorting, and then with OP9 stromal cells were cultured and purified endothelial cells marked by VE-cadherin (09-EC); high dose of trans retinoic acid (RA) induced iPS differentiation into smooth muscle cells. To detect the expression of gene differentiation cells. The marker and cell immune function, compared with normal mouse embryonic stem cell differentiation efficiency.
Results: the mouse iPS cells to endothelial cells and smooth muscle cell differentiation, differentiation efficiency and normal mouse embryonic stem cells are similar. The differentiation of endothelial cells can express CD31, CD144 and other surface markers, swallow DiI-Ac-LDL, and can form blood vessels in Matrigel. Smooth muscle cell differentiation could express SMAa, SMMHC and so on, have systolic function in the carbachol stimulation. Increased expression of endothelial or smooth muscle cell specific genes during the differentiation of Oct4, Sox2, Klf4 and c-Myc expression in iPS cells induced by the formation of these four transcription factors were significantly reduced.
Conclusion: mouse iPS cells can induce and differentiate into endothelial cells and smooth muscle cells, and their differentiation potential is similar to that of mouse embryonic stem cells.
Screening of synergistic transcription factors of the important regulatory factor Myocardin of smooth muscle cells in the fourth chapter
Objective: the proliferation and differentiation of vascular intimal hyperplasia of smooth muscle cells, coronary artery stenosis and vascular pathology are closely related, Myocardin is a transcription factor essential to smooth muscle differentiation in the process of the experiment, hope through the combination of screening and Myocardin protein found important transcription factor in new smooth muscle differentiation process or transcription factor, research in the process of smooth muscle differentiation and function of the signaling pathway, and a deeper understanding of the molecular mechanism of smooth muscle differentiation.
Methods: screening can upregulate the expression of Myocardin transcription factor 1170 transcription factor by luciferase activity assay, and co immunoprecipitation method to further confirm the physical candidate factors and the levels of Myocardin protein binding by bioinformatics analysis methods such as screening of candidate factors, to determine its role in regulating the differentiation of smooth muscle cell differentiation the model of smooth muscle cell in mouse embryonic stem cells.
Results: We screened ZNF297B, RAI14 and MAGED1 transcription factors from 1170 transcription factors, determined that they were highly expressed in smooth muscle cells, upregulated during smooth muscle differentiation, and could directly bind to Myocardin, and their over expression could promote the expression of smooth muscle differentiation genes.
Conclusion: the transcription factors such as ZNF297B, RAI14 and MAGED1 may be important transcriptional factors regulating the differentiation of smooth muscle cells.
Fifth chapter zinc finger protein 297B synergistic Myocardin to regulate the differentiation of smooth muscle cells
Objective: To investigate the expression and mechanism of zinc finger protein 297B (ZNF297B) during the differentiation of smooth muscle cells.
Methods: using quantitative real-time PCR detection of ZNF297B in human, rat, expression of smooth muscle cells in mice in vivo and in vitro and in vivo and in vitro differentiation or proliferation of smooth muscle cells in expression; plasmid ZNF297B-Myc was constructed and the distribution of immunofluorescence to detect the expression of ZNF297B-Myc in rat smooth muscle cells; combined detection of ZNF297B and experiment Myocardin interaction and co precipitating with luciferase activity detection and immunity.
Results: high specific expression of ZNF297B in smooth muscle cells, expression of differentiation into smooth muscle cells in mouse embryonic stem cells, down regulated in smooth muscle cell proliferation stimulated by PDGFBB process, the first increase after the decline of.ZNF297B mainly distributed in the nucleus and in the perinuclear cytoplasmic.ZNF297B can bind with Myocardin protein expression in the intima of carotid artery balloon injury the rats in the repair process, luciferase activity assay showed that the expression of ZNF297B is regulated by Myocardin.
Conclusion: ZNF297B may play an important role in the regulation of smooth muscle cell differentiation. This effect may be achieved through the synergistic effect of ZNF297B and Myocardin.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

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