本文選題：血液血管干細胞　切入點：BL-CFC　出處：《廣州醫(yī)學(xué)院》2010年碩士論文

【摘要】：血液血管干細胞(hemangioblast)是一種具有雙向分化潛能的干細胞,在體內(nèi)外發(fā)育的過程中,能夠分化成為造血細胞和內(nèi)皮細胞。血液血管干細理論已經(jīng)通過在胚胎干細胞分化模型中得到驗證,即胚胎干細胞經(jīng)2.5-3.5天分化為擬胚體,再在特殊的分化體系中可以形成一種特殊的Blast樣集落,其具有同時分化產(chǎn)生內(nèi)皮細胞和造血細胞的能力,這種形成Blast樣集落的細胞被稱為BL-CFC(the blast colony-forming cell)。近年來,血液血管干細胞在起源、分子生物學(xué)特點、體外誘導(dǎo)分化等方面的研究取得了較大進展。既往研究發(fā)現(xiàn)小鼠胚胎定向造血的起源部位AGM區(qū)亦存在類似血液血管干細胞的前體HPP-HA。之后在小鼠胚胎AGM區(qū)同樣發(fā)現(xiàn)了BL-CFC的存在,建立了特殊的的血液血管干細胞模型,此模型已被應(yīng)用于研究小鼠AGM區(qū)血液血管干細胞的發(fā)育和調(diào)控,如經(jīng)典的造血生長因子IL-3也可以促進血液血管干細胞的發(fā)育和分化。然而,鑒于血清培養(yǎng)體系營養(yǎng)成分復(fù)雜,不利于精細研究某些細胞因子對血液血管干細胞的真實作用。因此,本研究在小鼠胚胎AGM區(qū)已建立的BL-CFC模型的基礎(chǔ)上,建立了一個BL-CFC的無血清孵育培養(yǎng)模型,優(yōu)化了常規(guī)的BL-CFC培養(yǎng)體系,并在此基礎(chǔ)上,研究造血發(fā)育相關(guān)因子對小鼠AGM區(qū)血液血管干細胞的調(diào)節(jié)作用。本研究首先在小鼠胚胎AGM區(qū)建立了BL-CFC的無血清孵育培養(yǎng)模型。取E10.5小鼠胚胎AGM區(qū),消化成單細胞,在無血清培養(yǎng)體系下孵育12h后進行半固體集落培養(yǎng)。在bFGF、SCF、VEGF、IL-6、IGF-I和LIF細胞生長因子的共同作用下培養(yǎng)3-3.5天后,可見一種特殊形態(tài)的集落開始出現(xiàn),其形態(tài)和分化特征與E9.5-12.5小鼠胚胎AGM區(qū)來源的Blast集落相似。然后進行BL-CFC造血及內(nèi)皮分化功能鑒定。集落培養(yǎng)4-5天后,挑取單個集落進行造血集落培養(yǎng)。所有的BL-CFC可產(chǎn)生CFU-E、CFU-GM、CFU-Mix等一種或多種造血集落。貼壁細胞換EGM2內(nèi)皮細胞完全培養(yǎng)基繼續(xù)進行誘導(dǎo),第7-10天時可見貼壁細胞中有大量鵝卵石樣內(nèi)皮細胞以及少量成熟的造血細胞。隨后進行體外成血管功能檢測,結(jié)果表明:貼壁細胞高比例的吞噬LDL;體外Matrigel上可形成血管樣結(jié)構(gòu),呈網(wǎng)絡(luò)狀生長。同時,將BL-CFC在OP9基質(zhì)細胞上進行造血和內(nèi)皮細胞分化功能檢測。挑取單個Blast集落,接種于OP9基質(zhì)細胞上培養(yǎng)7-10天后,所有集落在OP9上均顯示造血細胞的分化潛能(CD45陽性),30%的HA在OP9上可以形成CD31陽性的管樣結(jié)構(gòu)。 接下來,研究了三種與造血發(fā)育相關(guān)的細胞因子IL-3、SCF和NGF對小鼠胚胎AGM區(qū)細胞的調(diào)節(jié)作用。將上述3種細胞因子與AGM區(qū)細胞分別在SR及FBS體系下體外共孵育12小時后進行BL-CFC接種,結(jié)果顯示IL-3在兩種體系下均能擴增BL-CFC,而NGF在兩種體系下對BL-CFC均無作用,SCF只在SR體系中對BL-CFC有明顯的擴增作用。 綜上,本研究在小鼠胚胎AGM區(qū)建立了BL-CFC的無血清孵育培養(yǎng)模型,優(yōu)化了常規(guī)的BL-CFC培養(yǎng)體系,基于上述模型,深入探討了造血發(fā)育相關(guān)因子IL-3、SCF和NGF在血清及無血清兩種孵育培養(yǎng)模式下對小鼠胚胎AGM區(qū)細胞的調(diào)節(jié)作用,與血清體系相比,無血清體系更能靈敏、準(zhǔn)確的反映細胞因子對血液血管干細胞的作用,從而體現(xiàn)了無血清培養(yǎng)模型的優(yōu)勢。
[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.

【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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相關(guān)期刊論文 前1條

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本文編號：1696716