本文關(guān)鍵詞：胚胎干細(xì)胞或胚胎樣干細(xì)胞向血管細(xì)胞的分化及調(diào)控機(jī)制研究　出處：《中南大學(xué)》2010年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 內(nèi)皮細(xì)胞 平滑肌細(xì)胞 胚胎干細(xì)胞 iPS細(xì)胞 分化 轉(zhuǎn)錄因子 鋅指蛋白297B

【摘要】： 血管系統(tǒng)是人類最大,分布最廣的系統(tǒng)。血管細(xì)胞主要包括內(nèi)皮細(xì)胞和平滑肌細(xì)胞。血管細(xì)胞的功能異�？梢詫�(dǎo)致動(dòng)脈粥樣硬化,心肌梗塞,腦中風(fēng)等血管疾病。 胚胎干細(xì)胞來源于囊胚內(nèi)細(xì)胞團(tuán),是具有無(wú)限增殖能力和向各胚層細(xì)胞分化能力的全能干細(xì)胞。胚胎干細(xì)胞向內(nèi)皮細(xì)胞或平滑肌細(xì)胞分化的研究對(duì)血管細(xì)胞的發(fā)育生物學(xué),心血管藥物的篩選,并可望為細(xì)胞移植治療提供細(xì)胞來源。本研究論文主要進(jìn)行小鼠和人類的胚胎干細(xì)胞(ESCs)及胚胎干細(xì)胞樣細(xì)胞(iPS細(xì)胞)向內(nèi)皮細(xì)胞(ECs)和平滑肌細(xì)胞(SMCs)分化的體系構(gòu)建及相關(guān)機(jī)制研究,包括以下五個(gè)方面：1、小鼠骨髓內(nèi)皮細(xì)胞條件培養(yǎng)液對(duì)小鼠胚胎干細(xì)胞向內(nèi)皮細(xì)胞分化的誘導(dǎo)作用；2、人類胚胎干細(xì)胞來源的內(nèi)皮樣細(xì)胞與臍血來源的內(nèi)皮祖細(xì)胞和臍靜脈內(nèi)皮細(xì)胞的比較；3、iPS細(xì)胞向內(nèi)皮細(xì)胞及平滑肌細(xì)胞的分化；4、平滑肌細(xì)胞分化過程中促進(jìn)分化的轉(zhuǎn)錄因子的篩選及調(diào)控機(jī)制的初步研究；5、鋅指蛋白297B協(xié)同Myocardin調(diào)節(jié)平滑肌細(xì)胞的分化。 第一章小鼠骨髓內(nèi)皮細(xì)胞條件培養(yǎng)液促進(jìn)鼠胚胎干細(xì)胞向內(nèi)皮細(xì)胞的誘導(dǎo)分化 目的：小鼠骨髓內(nèi)皮細(xì)胞的條件培養(yǎng)液在以前的實(shí)驗(yàn)中被證明能促進(jìn)造血細(xì)胞的增殖及分化,促進(jìn)骨髓內(nèi)皮細(xì)胞的生長(zhǎng)。本實(shí)驗(yàn)研究?jī)?nèi)皮細(xì)胞條件培養(yǎng)基(mEC-CM)對(duì)小鼠胚胎干細(xì)胞向內(nèi)皮細(xì)胞分化的促進(jìn)作用及分化的內(nèi)皮細(xì)胞的純化。 方法：在這個(gè)研究中,我們用mEC-CM誘導(dǎo)mESCs向內(nèi)皮細(xì)胞前體細(xì)胞Flkl+細(xì)胞分化,并將誘導(dǎo)分化的效率同細(xì)胞因子(VEGF,EGF,bFGF和IGF-1)誘導(dǎo)的效率相比較。我們還從分化的mESCs中機(jī)械挑選了吞噬DiI-Ac-LDL的鵝卵石樣細(xì)胞,分析其生物學(xué)特性。 結(jié)果：mEC-CM能明顯促進(jìn)mESCs向Flkl+細(xì)胞的分化,其效率與細(xì)胞因子的誘導(dǎo)相似,mEC-CM與細(xì)胞因子組合沒有協(xié)同誘導(dǎo)作用。mEC-CM的誘導(dǎo)結(jié)合機(jī)械挑選DiI-Ac-LDL標(biāo)記陽(yáng)性的鵝卵石樣細(xì)胞的方法,能純化分化的內(nèi)皮樣細(xì)胞,表達(dá)內(nèi)皮細(xì)胞標(biāo)志,結(jié)合UEA1,并能在體外形成血管樣結(jié)構(gòu)。 結(jié)論：mEC-CM能誘導(dǎo)mESCs向內(nèi)皮細(xì)胞的分化。機(jī)械挑選DiI-Ac-LDL標(biāo)記陽(yáng)性的鵝卵石樣細(xì)胞的方法能純化mESCs分化的內(nèi)皮樣細(xì)胞。 第二章胚胎干細(xì)胞來源的內(nèi)皮細(xì)胞的誘導(dǎo)分化及其與人臍靜脈內(nèi)皮細(xì)胞和臍血內(nèi)皮祖細(xì)胞的比較 目的：從人類胚胎干細(xì)胞分化系統(tǒng)中分選出內(nèi)皮前體細(xì)胞(KDR+細(xì)胞),研究其特性,并與臍血來源的內(nèi)皮祖細(xì)胞和臍靜脈內(nèi)皮細(xì)胞進(jìn)行比較。 方法：用本研究所建立的人類胚胎干細(xì)胞株chESC-1, chESC-3, chESC-8, chESC-20和chESC-22,自發(fā)分化成9天的擬胚體,磁珠分選出KDR+細(xì)胞,在內(nèi)皮細(xì)胞培養(yǎng)基培養(yǎng)后,檢測(cè)其內(nèi)皮細(xì)胞標(biāo)志的表達(dá),生長(zhǎng)特性,及其內(nèi)皮細(xì)胞功能,并與臍血內(nèi)皮祖細(xì)胞和臍靜脈內(nèi)皮細(xì)胞進(jìn)行比較。 結(jié)果：從9天EBs中分選出KDR+細(xì)胞在內(nèi)皮細(xì)胞培養(yǎng)基中培養(yǎng)后,與成體內(nèi)皮細(xì)胞相比較,胚胎干細(xì)胞來源的KDR+細(xì)胞表達(dá)更多的內(nèi)皮祖細(xì)胞標(biāo)志如CD133,CD34,不表達(dá)成熟內(nèi)皮細(xì)胞標(biāo)志vWF,能結(jié)合UEA1,吞噬DiI-Ac-LDL,并能在matrigel上形成血管樣結(jié)構(gòu)。在體外培養(yǎng)過程中,KDR+細(xì)胞的祖細(xì)胞標(biāo)志逐漸減少,出現(xiàn)成熟內(nèi)皮細(xì)胞標(biāo)志。 結(jié)論：人胚胎干細(xì)胞株能向內(nèi)皮細(xì)胞分化,分選得到的KDR+細(xì)胞在內(nèi)皮細(xì)胞培養(yǎng)條件下與成體內(nèi)皮細(xì)胞比較顯示其內(nèi)皮祖細(xì)胞特性,并能在體外培養(yǎng)中向成熟內(nèi)皮細(xì)胞分化。 第三章iPS細(xì)胞向內(nèi)皮細(xì)胞和平滑肌細(xì)胞的誘導(dǎo)分化 目的：成纖維細(xì)胞轉(zhuǎn)染Oct4, c-Myc, Sox2和Klf4四種因子得到的誘導(dǎo)的多能干細(xì)胞(induced pluripotent stem cells,iPS)是一種胚胎干細(xì)胞樣細(xì)胞,能無(wú)限增殖并具有多向分化潛能,較之胚胎干細(xì)胞,iPS細(xì)胞避免了倫理問題,且能建立患者特異性的細(xì)胞株,具有很高的臨床應(yīng)用價(jià)值。本研究探討小鼠iPS細(xì)胞向血管細(xì)胞(內(nèi)皮細(xì)胞和平滑肌細(xì)胞)分化的能力。 方法：采用單層貼壁誘導(dǎo)及流式細(xì)胞術(shù)進(jìn)行Flkl+細(xì)胞分化與分選,進(jìn)而與OP9基質(zhì)細(xì)胞共培養(yǎng),并用VE-cadherin為標(biāo)志純化內(nèi)皮細(xì)胞(09-EC)；用高劑量反式視黃酸(RA)誘導(dǎo)iPS向平滑肌細(xì)胞分化。檢測(cè)分化細(xì)胞的基因表達(dá),免疫標(biāo)記和細(xì)胞功能,與普通小鼠胚胎干細(xì)胞的分化效率相比較。 結(jié)果：小鼠iPS細(xì)胞可以向內(nèi)皮細(xì)胞和平滑肌細(xì)胞分化,分化效率與普通小鼠胚胎干細(xì)胞相似。分化的內(nèi)皮細(xì)胞能表達(dá)CD31,CD144等表面標(biāo)志,吞噬DiI-Ac-LDL,并能在matrigel上形成血管。分化的平滑肌細(xì)胞能表達(dá)SMAa, SMMHC等標(biāo)志,在碳酰膽堿刺激下有收縮功能。分化過程中內(nèi)皮或平滑肌細(xì)胞特異性基因表達(dá)上調(diào),而Oct4、Sox2、Klf4和c-Myc這四種誘導(dǎo)iPS細(xì)胞形成的轉(zhuǎn)錄因子的表達(dá)顯著下調(diào)。 結(jié)論：小鼠iPS細(xì)胞能向內(nèi)皮細(xì)胞和平滑肌細(xì)胞誘導(dǎo)分化,其分化潛能與小鼠胚胎干細(xì)胞相似。 第四章平滑肌細(xì)胞重要調(diào)節(jié)因子Myocardin的協(xié)同轉(zhuǎn)錄因子的篩選 目的：平滑肌細(xì)胞的增殖分化與血管內(nèi)膜增生,冠狀動(dòng)脈狹窄等血管病理密切相關(guān),Myocardin是平滑肌分化過程中不可缺少的一個(gè)協(xié)同轉(zhuǎn)錄因子,本實(shí)驗(yàn)希望通過篩選與Myocardin結(jié)合的蛋白,找到在平滑肌分化過程中新的重要的轉(zhuǎn)錄因子或協(xié)同轉(zhuǎn)錄因子,研究其在平滑肌分化過程中的功能及所屬信號(hào)通路,進(jìn)而更深入了解平滑肌分化的分子機(jī)制。 方法：用熒光素酶活性實(shí)驗(yàn)篩選1170個(gè)轉(zhuǎn)錄因子中能上調(diào)Myocardin表達(dá)的轉(zhuǎn)錄因子,并用免疫共沉淀等方法進(jìn)一步確認(rèn)候選因子與Myocardin蛋白水平的物理結(jié)合,通過生物信息學(xué)分析等方法篩選出候選因子,在小鼠胚胎干細(xì)胞向平滑肌細(xì)胞分化模型中確定其在平滑肌細(xì)胞分化中的調(diào)節(jié)作用。 結(jié)果：我們從1170個(gè)轉(zhuǎn)錄因子中篩選出ZNF297B,RAI14和MAGED1等轉(zhuǎn)錄因子,確定其在平滑肌中高表達(dá),在平滑肌分化過程中上調(diào),并能與Myocardin直接結(jié)合,其過表達(dá)能促進(jìn)平滑肌分化基因的表達(dá)。 結(jié)論：ZNF297B,RAI14和MAGED1等轉(zhuǎn)錄因子可能是調(diào)節(jié)平滑肌分化的重要轉(zhuǎn)錄因子。 第五章鋅指蛋白297B協(xié)同Myocardin調(diào)節(jié)平滑肌細(xì)胞的分化 目的：探討鋅指蛋白297B(ZNF297B)在平滑肌細(xì)胞分化過程中的表達(dá)及相關(guān)機(jī)制。 方法：用定量實(shí)時(shí)PCR檢測(cè)ZNF297B在人,大鼠,小鼠平滑肌細(xì)胞中體內(nèi)／體外的表達(dá)量及在平滑肌細(xì)胞體內(nèi)和體外分化或增殖過程中的表達(dá)變化；構(gòu)建ZNF297B-Myc表達(dá)質(zhì)粒,免疫熒光檢測(cè)ZNF297B-Myc在大鼠平滑肌細(xì)胞中的表達(dá)分布；用熒光素酶活性檢測(cè)及免疫共沉淀實(shí)驗(yàn)檢測(cè)ZNF297B與Myocardin的結(jié)合及相互作用。 結(jié)果：ZNF297B在平滑肌細(xì)胞中特異性高表達(dá),在小鼠胚胎干細(xì)胞向平滑肌細(xì)胞分化過程中表達(dá)上調(diào),在PDGFBB刺激的平滑肌細(xì)胞增殖過程中下調(diào),在大鼠頸動(dòng)脈球囊損傷內(nèi)膜修復(fù)過程中表達(dá)先上調(diào)后下降。ZNF297B主要分布于細(xì)胞核及核周的細(xì)胞質(zhì)。ZNF297B能與Myocardin蛋白結(jié)合,熒光素酶活性檢測(cè)顯示ZNF297B的表達(dá)受Myocardin的調(diào)控。 結(jié)論：ZNF297B可能在平滑肌細(xì)胞分化過程中起到重要正向調(diào)控作用,這一作用很可能是通過ZNF297B與Myocardin的協(xié)同作用來完成。
[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.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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