造血发育相关因子对小鼠AGM区血液血管干细胞的调节作用

发布时间：2018-04-01 18:21

本文选题：血液血管干细胞　切入点：BL-CFC　出处：《广州医学院》2010年硕士论文

【摘要】：血液血管干细胞(hemangioblast)是一种具有双向分化潜能的干细胞,在体内外发育的过程中,能够分化成为造血细胞和内皮细胞。血液血管干细理论已经通过在胚胎干细胞分化模型中得到验证,即胚胎干细胞经2.5-3.5天分化为拟胚体,再在特殊的分化体系中可以形成一种特殊的Blast样集落,其具有同时分化产生内皮细胞和造血细胞的能力,这种形成Blast样集落的细胞被称为BL-CFC(the blast colony-forming cell)。近年来,血液血管干细胞在起源、分子生物学特点、体外诱导分化等方面的研究取得了较大进展。既往研究发现小鼠胚胎定向造血的起源部位AGM区亦存在类似血液血管干细胞的前体HPP-HA。之后在小鼠胚胎AGM区同样发现了BL-CFC的存在,建立了特殊的的血液血管干细胞模型,此模型已被应用于研究小鼠AGM区血液血管干细胞的发育和调控,如经典的造血生长因子IL-3也可以促进血液血管干细胞的发育和分化。然而,鉴于血清培养体系营养成分复杂,不利于精细研究某些细胞因子对血液血管干细胞的真实作用。因此,本研究在小鼠胚胎AGM区已建立的BL-CFC模型的基础上,建立了一个BL-CFC的无血清孵育培养模型,优化了常规的BL-CFC培养体系,并在此基础上,研究造血发育相关因子对小鼠AGM区血液血管干细胞的调节作用。本研究首先在小鼠胚胎AGM区建立了BL-CFC的无血清孵育培养模型。取E10.5小鼠胚胎AGM区,消化成单细胞,在无血清培养体系下孵育12h后进行半固体集落培养。在bFGF、SCF、VEGF、IL-6、IGF-I和LIF细胞生长因子的共同作用下培养3-3.5天后,可见一种特殊形态的集落开始出现,其形态和分化特征与E9.5-12.5小鼠胚胎AGM区来源的Blast集落相似。然后进行BL-CFC造血及内皮分化功能鉴定。集落培养4-5天后,挑取单个集落进行造血集落培养。所有的BL-CFC可产生CFU-E、CFU-GM、CFU-Mix等一种或多种造血集落。贴壁细胞换EGM2内皮细胞完全培养基继续进行诱导,第7-10天时可见贴壁细胞中有大量鹅卵石样内皮细胞以及少量成熟的造血细胞。随后进行体外成血管功能检测,结果表明:贴壁细胞高比例的吞噬LDL;体外Matrigel上可形成血管样结构,呈网络状生长。同时,将BL-CFC在OP9基质细胞上进行造血和内皮细胞分化功能检测。挑取单个Blast集落,接种于OP9基质细胞上培养7-10天后,所有集落在OP9上均显示造血细胞的分化潜能(CD45阳性),30%的HA在OP9上可以形成CD31阳性的管样结构。接下来,研究了三种与造血发育相关的细胞因子IL-3、SCF和NGF对小鼠胚胎AGM区细胞的调节作用。将上述3种细胞因子与AGM区细胞分别在SR及FBS体系下体外共孵育12小时后进行BL-CFC接种,结果显示IL-3在两种体系下均能扩增BL-CFC,而NGF在两种体系下对BL-CFC均无作用,SCF只在SR体系中对BL-CFC有明显的扩增作用。综上,本研究在小鼠胚胎AGM区建立了BL-CFC的无血清孵育培养模型,优化了常规的BL-CFC培养体系,基于上述模型,深入探讨了造血发育相关因子IL-3、SCF和NGF在血清及无血清两种孵育培养模式下对小鼠胚胎AGM区细胞的调节作用,与血清体系相比,无血清体系更能灵敏、准确的反映细胞因子对血液血管干细胞的作用,从而体现了无血清培养模型的优势。
[Abstract]:The blood vessel stem cell (Hemangioblast) is a kind of bipotential stem cells during development in vitro and in vivo, can differentiate into hematopoietic cells and endothelial cells. The Hemangioblast theory has been verified by the embryonic stem cell differentiation model, namely embryonic stem cells after 2.5-3.5 days of differentiation into embryoid bodies then, you can form a special kind of Blast colony in particular differentiation system, it also has the ability to differentiate into endothelial cells and hematopoietic cells, the formation of Blast like colonies of cells called BL-CFC (the blast colony-forming cell). In recent years, the blood vessels in the stem cell origin, molecular biological characteristics that research has made great progress in vitro differentiation and other aspects. Previous studies have found that the site of origin of AGM mouse embryonic hematopoietic region are similar to the blood vascular stem cell precursor HPP-HA. In the mouse AGM region also found the existence of BL-CFC, established a special blood vascular stem cell model, this model has been applied to the study of mouse AGM blood vessel stem cell development and regulation, such as the classical hematopoietic growth factor IL-3 can also promote cell development and differentiation of blood vessel stem. However, in view of the serum culture system has complex nutrients, is not conducive to the detailed study of some cytokines stem cells of blood vessels is true. Therefore, this study has established the foundation of BL-CFC model in mouse AGM region on the establishment of a serum-free incubation culture model of BL-CFC, optimize the conventional BL-CFC culture system, and based on the study of developmental hematopoiesis related cytokines regulating effects on Hemangioblast in mouse AGM region. In this paper, the mouse AGM region established serum-free Fu Yupei raising model of BL-CFC E10.5. The mouse AGM region, digest it into single cells in culture system after 12h incubation of semi-solid colony culture without serum. In bFGF, SCF, VEGF, IL-6, 3-3.5 and IGF-I together after cultured LIF cell growth factor, that is a special form of the colony began to appear. The morphology and differentiation characteristics and E9.5-12.5 mouse AGM region Blast from the colony is similar. Then BL-CFC hematopoietic and endothelial differentiation. Identification of colony after 4-5 days of culture, from single colony culture hematopoietic colony. All BL-CFC can produce CFU-E, CFU-GM, CFU-Mix and one or more hematopoietic colony with. Cell wall in EGM2 endothelial cells to complete medium for induction, 7-10 days with a large number of visible cobblestone like endothelial cells and several mature hematopoietic cells wall cells. Then vascular function in vitro detection, the results show that the thin wall A high percentage of phagocytosis of LDL cells in vitro; Matrigel can form vascular like structure, showed a net growth. At the same time, the BL-CFC of hematopoietic and endothelial cell differentiation function test in OP9 stromal cells. From single colony Blast, inoculated in OP9 stromal cells cultured for 7-10 days, all the colonies in OP9 showed differentiation the potential of hematopoietic cells (CD45 positive), 30% HA in OP9 form CD31 positive tube like structure.
Then, three kinds of cytokines related to IL-3 development and hematopoiesis, regulation of SCF and NGF on mouse embryonic AGM cells. These 3 kinds of cytokines and AGM cells respectively in SR and FBS system in vitro were incubated for 12 hours after BL-CFC inoculation, the results showed that IL-3 could be amplified in BL-CFC the two systems, and NGF in the two systems had no effect on BL-CFC, SCF only in the SR system of BL-CFC amplification effect.
In summary, this study in the mouse AGM region established serum-free incubation culture model of BL-CFC, optimize the conventional BL-CFC culture system, based on the above model, discusses the developmental hematopoiesis related cytokines IL-3, SCF and NGF two kinds of serum free incubation on regulation of mouse embryonic AGM cells model in serum and, compared with serum system, serum free system is more sensitive and accurate reflection of cytokine stem cells on blood vessels, which reflects the serum-free culture model.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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