人胚胎干细胞向血管内皮细胞的诱导分化及后者迁移机制的研究

发布时间：2018-03-28 17:33

本文选题：人胚胎干细胞　切入点：定向分化　出处：《复旦大学》2006年博士论文

【摘要】： 人胚胎干细胞向血管内皮细胞的诱导分化及后者迁移机制的研究目的：探讨人胚胎干细胞(hESC)向造血-内皮系诱导分化的能力，将hESC定向分化为高纯度的hESC来源内皮细胞，为hESC应用于构建组织工程血管提供依据。以hESC作为研究载体，建立模仿人胚胎发育过程中血管生成的芽生EB模型，探究SDF-1/CXCR4轴对人类胚胎干细胞来源内皮细胞的迁移和血管生成的影响。材料与方法：1．选用人胚胎干细胞系(NIH人胚胎干细胞登记署登记号为：WA01和09，简称为H1、H9)为研究对象。将未分化hESC培养在经丝裂霉素或照射灭活的小鼠成纤维细胞饲养层上，每日换液一次，第5-7天后进行细胞传代，检测细胞表面期特异性抗原SSEA(Stage specific embryonic antigen)-1、SSEA-4、TRA(tumor rejection antigen)-1-60、TRA-1-81的表达以明确细胞是否属于未分化状态。2．以与小鼠骨髓基质细胞(OP9)共培养或以悬浮类胚体(embryo body，EB)形式进行造血-内皮系诱导分化，每2-3日换液一次。检测分化过程中细胞表面SSEA-1、SSEA-4、和造血-内皮系转录调控基因(GATA-1、SCL、LMO-2、CD31、Tie-2)、以及该二系细胞表面标志的变化，并以半固体培养基进行造血集落的培养。3．以磁珠分选分化后H1来源CD34~+细胞，将这部分细胞置于内皮培养体系再诱导，获取一群类似于成人脐静脉内皮细胞的贴壁细胞，并对这部分细胞进行内皮细胞的性质鉴定，包括CD31、CD34、Flk-1、VE-Cadherin和vWF等表面标记的表达、吞噬LDL的能力以及在Matrigel基质表面形成网状结构的能力等。4．将分化后EB置于胶原基质和VEGF、FGF等血管生成因子的混合体系中，进行芽生EB的诱导，摸索该体系的最佳培养条件，包括EB的时龄、培养的时间、VEGF/FGF的浓度组合等，并进行芽生结构内皮系性质的鉴定，如：CD31荧光标记和血管生成抑制因子对芽状结构的抑制作用等。5．以RT-PCR法和流式细胞仪检测H1分化不同阶段、H1来源内皮细胞中CXCR4和SDF-1的表达水平。将20μl hESC来源内皮细胞置于微孔膜一侧，微孔膜另一侧加入不同浓度梯度的SDF-1，以ChemoTx系统进行H1来源内皮细胞向SDF-1的跨膜迁移能力检测。以外源性SDF-1上调SDF-1/CXCR4轴、或以AMD3100、CXCR4抗体阻断SDF-1/CXCR4的相互结合，观察这些调节作用对H1来源内皮细胞迁移能力、以及hESC来源原始血管生成的影响。结果：1．未分化hESC集落表现为圆形或椭圆形、平坦、细胞排列紧密的细胞团，表达SSEA-4、TRA-1-60、TRA-1-81等未分化hESC的特异性标志，，但不表达SSEA-1。随着细胞分化程度的提高，SSEA-4的表达逐渐减弱，而SSEA-1的表达进行性升高。2．HESC与小鼠骨髓基质细胞共培养或以EB形式诱导分化后，原本在未分化状态时不表达的造血-内皮系基因在RT-PCR检测时逐渐显示阳性条带，而未分化基因Oct-4的表达逐渐减弱，中胚层标志基因Brachyury在分化后第6天左右消失。VEGF-R2(Flk-1)在未分化hESC中也有一定程度的表达，随着分化程度的提高，Flk-1的表达程度也逐渐上升。3．细胞在分化后开始表达CD34这一造血/内皮系共同标记，且在第12天左右达到峰值，同期也获得了最多的造血集落数。此后，CD34的阳性率和造血集落的数量逐渐减弱，但粒-巨噬系集落占所有造血集落的比例却逐渐上升。4．我们选择分化后第10天-12天这一CD34表达高峰期为分选时机，以磁珠分选的方法分选hESC来源CD34~+细胞，CD34~+细胞的纯度达到90％以上，这部分细胞同时还表达高水平的CD31、但不表达CD45。将hESC来源CD34~+细胞在内皮细胞培养体系中进一步分化成熟，所获得的贴壁细胞形态类似于成熟人脐静脉内皮细胞，具有成熟内皮细胞的特征，表达高水平的CD31、CD34、Flk-1、VE-Cadherin和vWF，能吞噬Dil-AcLDL，在Matrigel基质表面形成网络状结构。5．将分化第11天的EB置于含有VEGF、FGF的胶原基质中，从EB中可以伸展出有内皮细胞积聚的、类似于血管腔样结构的芽状分支，为芽生EB。芽生EB间还可形成网络状结构，是研究人类胚胎发育过程中血管生成的一种模型。6．HESC的各个分化阶段、hESC来源内皮细胞表面都表达有CXCR4，而且VEGF可以上调细胞表面CXCR4的表达水平。HESC来源内皮细胞可以顺SDF-1浓度梯度出现向SDF-1的跨膜迁移，SDF-1可以增强hESC来源内皮细胞的伸展和相互接触，促进由内皮细胞构成的网络状结构的形成。阻断SDF-1和CXCR4的相互作用可以破坏内皮细胞网络结构、抑制芽状结构的生长、切断芽生EB间的联系。结论：1．在小鼠胚胎成纤维细胞滋养层上，人胚胎干细胞系能够无限增殖并保持其未分化状态。2．在小鼠骨髓基质细胞表面或以悬浮EB形式，未分化hESC能够进入中胚层途径，向造血-内皮系进一步分化。分化后细胞逐渐表达造血/内皮系表面标记，SSEA-4、Oct-4等未分化标志的表达则明显减弱。3．分化后第12天左右，hESC来源的造血细胞大量涌现，随着分化程度的加深，细胞有逐步脱离原始胚胎造血阶段的可能。4．选择分化后第10天-12天这一CD34近高峰期为分选时机，可以获得高纯度的hESC来源CD34阳性细胞，而且这部分细胞可以进一步诱导分化为成熟的血管内皮细胞。因此，我们已经建立了一种将hESC 定向分化为成熟血管内皮细胞的方法。5．HESC来源内皮细胞表达有SDF-1/CXCR4这一趋化因子配体/受体对，SDF-1/CXCR4可以调节hESC来源内皮细胞的迁移能力。VEGF可能通过上调细胞表面CXCR4的表达而促进芽生EB的生成，与SDF-1/CXCR4共同介导人胚胎发生过程中原始血管网的形成。
[Abstract]:Differentiation of Human Embryonic Stem Cells into Vascular Endothelial Cells and Study of the Mechanism of Migration ;

Objective : To investigate the ability of human embryonic stem cells ( hESC ) to induce differentiation of human embryonic stem cells ( hESC ) into high - purity hESC - derived endothelial cells and to provide the basis for the application of hESC in the construction of tissue engineering vessels . Using hESC as the research carrier , an EB model of angiogenesis in human embryonic development was established , and the effects of SDF - 1 / CXCR4 axis on migration and angiogenesis of human embryonic stem cells derived from human embryonic stem cells were investigated .

Materials and Methods : 1 . Human embryonic stem cell line ( NIH human embryonic stem cell registration number : WA01 and 09 , abbreviated as H1 , H9 ) was used as the research object . Undifferentiated hESC was cultured on the feeder layer of mouse fibroblast feeder irradiated with mitomycin or irradiation . After 5 - 7 days , the expression of specific antigen SSEA ( Stage specific embryonic antigen ) -1 , SSEA - 4 , TRA ( tumor rejection antigen ) -1 - 60 , TRA - 1 - 81 was detected to determine whether the cell belonged to undifferentiated state . The cell surface SSEA - 1 , SSEA - 4 , and hematopoietic - endothelial transcription regulatory genes ( GATA - 1 , SCL , LMO - 2 , CD31 , Tie - 2 ) and the changes of surface markers of the two - line cells were detected in the differentiation process . CD34 + cells were isolated from human umbilical vein endothelial cells by magnetic beads . The cells were then cultured in endothelial culture system to obtain a group of adherent cells similar to adult human umbilical vein endothelial cells . The properties of these cells were identified , including the expression of surface markers such as CD31 , CD34 , Flk - 1 , VE - cadherin and VWF , the ability to phagocytize LDL and the ability to form reticular formation on the surface of Matrigel matrix . EB was induced by EB in the mixed system of angiogenic factors such as collagen matrix and VEGF and FGF . The optimal culture conditions of EB were determined , including the age of EB , the time of culture , the concentration of VEGF / FGF , and the identification of the properties of the endothelial system of the bud structure , such as the inhibitory effect of CD31 fluorescent label and angiogenesis inhibiting factor on the bud structure . The expression levels of CXCR4 and SDF - 1 in H1 - derived endothelial cells were detected by RT - PCR and flow cytometry .

Results : 1 . The expression of SSEA - 4 , TRA - 1 - 60 , TRA - 1 - 81 did not express SSEA - 1 . The expression of VEGF - R2 ( Flk - 1 ) in undifferentiated hESC was gradually decreased , and the expression of Flk - 1 increased gradually with the increase of differentiation degree . After differentiation , the CD34 + hematopoietic / endothelial cells were labeled and peaked at about 12 days , and the most hematopoietic colonies were obtained in the same period . After that , the positive rate of CD34 and the number of hematopoietic colonies decreased gradually , but the proportion of the granulocyte - macrophage colony to all hematopoietic colonies increased gradually . CD34 ~ + cells were isolated from human umbilical vein endothelial cells . The results showed that CD34 ~ + cells were mature in endothelial cells . The morphology of CD34 ~ + cells was similar to that of mature human umbilical vein endothelial cells . EB in the 11th day was placed in the collagen matrix containing VEGF and FGF . The accumulation of endothelial cells could be exhibited from EB . It was similar to the bud - like branch of the vascular cavity - like structure . It was also a model of angiogenesis in the development of human embryo . The expression level of CXCR4 is expressed on the endothelial cell surface of hESC , and the expression level of CXCR4 in the cell surface can be regulated by the VEGF . The migration of SDF - 1 to SDF - 1 can be enhanced by SDF - 1 . The interaction between SDF - 1 and CXCR4 can destroy the network structure of endothelial cells , inhibit the growth of bud - like structure , and cut off the relationship between budding and EB .

Conclusion : 1 . Human embryonic stem cells were able to proliferate and maintain their undifferentiated state on mouse embryonic fibroblast growth layers . In the mouse bone marrow stromal cell surface or in the form of suspension EB , the undifferentiated hESC was able to enter the mesoderm pathway to further differentiate into the hematopoietic - endothelium system . After differentiation , the cells gradually expressed hematopoietic / endothelial system surface markers , and the expression of SSEA - 4 , Oct - 4 and other undifferentiated markers decreased significantly . At the 12th day after differentiation , the hematopoietic cells of hESC origin are emerging , and with the deepening of differentiation degree , the cells are gradually separated from the possible hematopoietic stage of the original embryo . High - purity hESC - derived CD34 - positive cells can be obtained by selective differentiation from 10 to 12 days after differentiation , and these cells can be further induced to differentiate into mature vascular endothelial cells .

Directional differentiation into mature vascular endothelial cells . The expression of SDF - 1 / CXCR4 and SDF - 1 / CXCR4 in HESC - derived endothelial cells can regulate the migration of endothelial cells from hESC .

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329.2

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