【作者】 孙璇；

【导师】 卢光琇；

【作者基本信息】 中南大学 ， 干细胞工程学， 2010， 博士

【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。更多还原

【Abstract】 Vascular system is the biggest and the most widely distributed system in human being. Vascular cells include endothelial cells and smooth muscle cells. The disfunction of vascular cells can cause diseases such as atherosclerosis, myocardial infarction, stock and reginal ischemia.Embryonic stem cells (ESCs) are cell lines from inner cell mess of blastula. They can infinitely proliferate and are capable of differentiate into all the cell types of three layers. The in vitro system of differentiation of ESCs into vascular cells is a valuable tool to study vascular development, screen drugs for cardiovascular diseases, or potential cell transplantation in clinic applications. This thesis focuses on several issues that are related to the differentiation of ESCs or ESC-like cells (iPS) to endothelial cells and/or smooth muscle cells as well as the mechanisms of the differentiation, which include the following aspects:1. The promotional effects of conditional medium of endothelial cells to the differentiation of ESCs into endothelial cells; 2. The comparison of ESCs derived endothelial-like cells with adult endothelial progenitors and human umbilical cord vascular endothelial cells; 3. The differentiation of iPS cells into endothelial cells and smooth muscle cells; 4. The screening of the transcriptional factors that are related to smooth muscle differentiation and the mechanisms underneath; 5. ZNF297B regulates differentiation of smooth muscle cells through interaction with myocardin. Chapter 1 The effect of a conditional medium of endothelial cells in inducing the differentiation of mouse embryonic stem cells into endothelial cellsObjective:It has been proved that the conditional medium of mouse bone marrow endothelial cells (mEC-CM) could promote the proliferation and differentiation of embryonic or adult hematopoietic cells; it also could stimulate the growth of bone marrow endothelial cells. The purpose of this experiment is to study the effect of the cytokines in this mEC-CM in inducing the differentiation of mouse embryonic stem cells into endothelial cells.Methods:In this study, we induced the mESCs into endothelial progenitors with mEC-CM, and compared the differentiation efficiency with the induction by cytokine cocktail(VEGF,EGF,bFGF and IGF1). We also handpicked the DiI-Ac-LDL uptaken cells derived from mESCs and analyzed their characteristics. They were a pure cell population with endothelial characteristics.Results:mEC-CM could significantly promote the differentiation of mESCs into Flk1+ cells. The efficiency was similar to the induction by cytokine cocktail. We got pure cobblestone-like cell population by induction by mEC-CM combined with handpick-up Dil-Ac-LDL-positive cells. This cell population could express endothelial marker, bind UEA1, uptake DiI-Ac-LDL and form tube-like structure in vitro.Conclusions:mEC-CM can induce the differentiation of mESCs into endothelial cells. Manual selection of DiI-Ac-LDL-positive cells could be a potential method to isolate endothelial-like cells from differentiated mESCs.Chapter 2 Comparison the embryonic stem cell-derived KDR+ cells with cord blood endothelial progenitors and human umbilical cord vascular endothelial cellsObjective:To study the characteristics of human embryonic stem cell-derived endothelial progenitors (KDR+ cells), and compare them with CBEPCs and HUVECs.Methods:The human ES cell lines that established by our institute were allowed to spontaneously differentiated into 9 day embryoid bodies (9dEBs). Isolated the KDR+ cells from 9dEBs by MACS and cultured them in endothelial medium. Their expression of endothelial markers and their function were tested and compared with CBEPCs and HUVECs.Results:Compared with adult endothelial cells, the 9dEB-derived KDR+ cells expressed higher level of endothelial progenitor markers such as CD133 and CD34, showed endothelial function, but did not express the mature endothelial marker vWF. In the process of in vitro culture, the progenitor markers of KDR+ cells were downregulated, while mature endothelial markers were upregulated.Conclusions:The human ES cell lines established by our institute can differentiated into endothelial cells. The isolated KDR+ cells showed the endothelial progenitor characteristics and could differentiate into mature endothelial cells in vitro. Chapter 3 The induced differentiation of iPS cells into endothelial cells and smooth muscle cellsObjective:The induced pluripotent stem cells (iPS cells) in our experiment were derived from fibroblast cells that had been transfected by four transcriptional factors (Oct4,Nanog,Sox2 and Klf4). iPS is embryonic stem cell like pluripotent cell line. But compared with ESCs, iPS cell experiments avoid the ethical issues and make patient specific pluripotent cell line possible. This experiment studied the differentiation of iPS cells into vascular cells (endothelial cells and smooth muscle cells).Methods:09 iPS cells were induced to differentiate into endothelial cells by monolayer culture, FACS sorting Flk1+ cells, followed by OP9 stromal cell co-culture, and purified by FACS sorting VE-cadherin+ cells(09-EC). Smooth muscle cell differentiation of iPS cells was induced by high dose retinoid acid (RA).The specific marker expression and functional characteristics of the differentiated cells were analyzed. Gene expression during differentiation process were tested by real-time PCR. The differentiation efficiency was compared with ESC differentiation.Results:iPS cells could differentiate into endothelial cells and smooth muscle cells. The endothelial or smooth muscle specific genes were up-regulated in the differentiation process. Four reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) that induce the generation of iPS cells were significantly down-regulated in this process. The method and efficiency of differentiation were similar with normal mouse ESCs. Conclusions:This study supports that iPS cells can differentiate into endothelial cells and smooth muscle cells in vitro. The vascular differentiation ability of iPS cells is similar with ESCs This procedure may contribute to clinic application of patient-specific iPS cells derived cells in vascular degeneration diseases.Chapter 4 Screening of the co-activators of Myocardin—new discovery in mechanism of regulation of smooth muscle cell differentiationObjective:The proliferation and differentiation of smooth muscle cells is highly related to the mechanisms underneath angiopathological situation such as formation of neointima and coronary stenosis. Myocardin is a key factor in smooth muscle differentiation. In this experiment, we were looking for important transcriptional factors or co-activators in smooth muscle differentiation signaling pathway by screening the proteins that could bind and upregulate myocardin. By studying the function of the protein candidate, we could discover the molecular mechanism of smooth muscle differentiation.Methods:We discovered a list of candidates that could upregulate the expression of myocardin by screening 1170 transcriptional factor candidates by luciferase assay. After further confirmation of the binding between candidates and myocardin by co-IP and analysis with bioinformatics strategy, we got ZNF297B, RAI14 and MAGED1 as the most promising candidates.Results:ZNF297B, RAI14 and MAGED1 were highly expressed in smooth muscle cells and their expression level was upregulated during the differentiation of smooth muscle cells. They could bind to myocardin directly. The overexpression of these candidates promoted the differentiation of ESCs into smooth muscle cells.Conclusions:ZNF297B, RAI14 and MAGED1 could be important transcriptional factors to regulate smooth muscle differentiationChapter 5 ZNF297B regulates differentiation of smooth muscle cells through interaction with myocardinObjective:Discover the expression of ZNF297B in the differentiation of smooth muscle cells and investigate the related mechanism.Methods:The mRNA expression level of ZNF297B in human, rat and mouse smooth muscle cells and the change of ZNF297B expression level in smooth muscle cell differentiation and proliferation was tested by real-time PCR. The ZNF297B-Myc/His plasmid was constructed and transfected into rat smooth muscle cells to test the distribution of ZNF297B in cultured cells. The binding and interaction of ZNF297B and Myocardin was tested by luciferase assay and confirmed by co-immunoprecipitation.Results:ZNF297B was specificly highly expressed in smooth muscle cells. The expression level of ZNF297B was upregulated in the process of RA induced smooth muscle cell differentiation from mouse embryonic stem cells. ZNF297B expression was downregulated in PDGFBB stimulated smooth muscle cell proliferation process. In balloon-injured rat carotid arteries, ZNF297B was upregulated after 6 hours and then downregulated. ZNF297B mainly expressed in nucleus and the cytoplasm around nucleus. ZNF297B can bind and interact with Myocardin in protein level.Conclusions:ZNF297B may play an important role in smooth muscle cell differentiation. This function may related to the interaction between ZNF297B and Myocardin.更多还原