發(fā)布時間：2018-01-02 08:00

  本文關鍵詞：胚胎干細胞或胚胎樣干細胞向血管細胞的分化及調控機制研究　出處：《中南大學》2010年博士論文　論文類型：學位論文

  更多相關文章： 內皮細胞 平滑肌細胞 胚胎干細胞 iPS細胞 分化 轉錄因子 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)。血管細胞主要包括內皮細胞和平滑肌細胞。血管細胞的功能異�？梢詫е聞用}粥樣硬化,心肌梗塞,腦中風等血管疾病。 胚胎干細胞來源于囊胚內細胞團,是具有無限增殖能力和向各胚層細胞分化能力的全能干細胞。胚胎干細胞向內皮細胞或平滑肌細胞分化的研究對血管細胞的發(fā)育生物學,心血管藥物的篩選,并可望為細胞移植治療提供細胞來源。本研究論文主要進行小鼠和人類的胚胎干細胞(ESCs)及胚胎干細胞樣細胞(iPS細胞)向內皮細胞(ECs)和平滑肌細胞(SMCs)分化的體系構建及相關機制研究,包括以下五個方面：1、小鼠骨髓內皮細胞條件培養(yǎng)液對小鼠胚胎干細胞向內皮細胞分化的誘導作用；2、人類胚胎干細胞來源的內皮樣細胞與臍血來源的內皮祖細胞和臍靜脈內皮細胞的比較；3、iPS細胞向內皮細胞及平滑肌細胞的分化；4、平滑肌細胞分化過程中促進分化的轉錄因子的篩選及調控機制的初步研究；5、鋅指蛋白297B協同Myocardin調節(jié)平滑肌細胞的分化。 第一章小鼠骨髓內皮細胞條件培養(yǎng)液促進鼠胚胎干細胞向內皮細胞的誘導分化 目的：小鼠骨髓內皮細胞的條件培養(yǎng)液在以前的實驗中被證明能促進造血細胞的增殖及分化,促進骨髓內皮細胞的生長。本實驗研究內皮細胞條件培養(yǎng)基(mEC-CM)對小鼠胚胎干細胞向內皮細胞分化的促進作用及分化的內皮細胞的純化。 方法：在這個研究中,我們用mEC-CM誘導mESCs向內皮細胞前體細胞Flkl+細胞分化,并將誘導分化的效率同細胞因子(VEGF,EGF,bFGF和IGF-1)誘導的效率相比較。我們還從分化的mESCs中機械挑選了吞噬DiI-Ac-LDL的鵝卵石樣細胞,分析其生物學特性。 結果：mEC-CM能明顯促進mESCs向Flkl+細胞的分化,其效率與細胞因子的誘導相似,mEC-CM與細胞因子組合沒有協同誘導作用。mEC-CM的誘導結合機械挑選DiI-Ac-LDL標記陽性的鵝卵石樣細胞的方法,能純化分化的內皮樣細胞,表達內皮細胞標志,結合UEA1,并能在體外形成血管樣結構。 結論：mEC-CM能誘導mESCs向內皮細胞的分化。機械挑選DiI-Ac-LDL標記陽性的鵝卵石樣細胞的方法能純化mESCs分化的內皮樣細胞。 第二章胚胎干細胞來源的內皮細胞的誘導分化及其與人臍靜脈內皮細胞和臍血內皮祖細胞的比較 目的：從人類胚胎干細胞分化系統(tǒng)中分選出內皮前體細胞(KDR+細胞),研究其特性,并與臍血來源的內皮祖細胞和臍靜脈內皮細胞進行比較。 方法：用本研究所建立的人類胚胎干細胞株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+細胞,在內皮細胞培養(yǎng)基培養(yǎng)后,檢測其內皮細胞標志的表達,生長特性,及其內皮細胞功能,并與臍血內皮祖細胞和臍靜脈內皮細胞進行比較。 結果：從9天EBs中分選出KDR+細胞在內皮細胞培養(yǎng)基中培養(yǎng)后,與成體內皮細胞相比較,胚胎干細胞來源的KDR+細胞表達更多的內皮祖細胞標志如CD133,CD34,不表達成熟內皮細胞標志vWF,能結合UEA1,吞噬DiI-Ac-LDL,并能在matrigel上形成血管樣結構。在體外培養(yǎng)過程中,KDR+細胞的祖細胞標志逐漸減少,出現成熟內皮細胞標志。 結論：人胚胎干細胞株能向內皮細胞分化,分選得到的KDR+細胞在內皮細胞培養(yǎng)條件下與成體內皮細胞比較顯示其內皮祖細胞特性,并能在體外培養(yǎng)中向成熟內皮細胞分化。 第三章iPS細胞向內皮細胞和平滑肌細胞的誘導分化 目的：成纖維細胞轉染Oct4, c-Myc, Sox2和Klf4四種因子得到的誘導的多能干細胞(induced pluripotent stem cells,iPS)是一種胚胎干細胞樣細胞,能無限增殖并具有多向分化潛能,較之胚胎干細胞,iPS細胞避免了倫理問題,且能建立患者特異性的細胞株,具有很高的臨床應用價值。本研究探討小鼠iPS細胞向血管細胞(內皮細胞和平滑肌細胞)分化的能力。 方法：采用單層貼壁誘導及流式細胞術進行Flkl+細胞分化與分選,進而與OP9基質細胞共培養(yǎng),并用VE-cadherin為標志純化內皮細胞(09-EC)；用高劑量反式視黃酸(RA)誘導iPS向平滑肌細胞分化。檢測分化細胞的基因表達,免疫標記和細胞功能,與普通小鼠胚胎干細胞的分化效率相比較。 結果：小鼠iPS細胞可以向內皮細胞和平滑肌細胞分化,分化效率與普通小鼠胚胎干細胞相似。分化的內皮細胞能表達CD31,CD144等表面標志,吞噬DiI-Ac-LDL,并能在matrigel上形成血管。分化的平滑肌細胞能表達SMAa, SMMHC等標志,在碳酰膽堿刺激下有收縮功能。分化過程中內皮或平滑肌細胞特異性基因表達上調,而Oct4、Sox2、Klf4和c-Myc這四種誘導iPS細胞形成的轉錄因子的表達顯著下調。 結論：小鼠iPS細胞能向內皮細胞和平滑肌細胞誘導分化,其分化潛能與小鼠胚胎干細胞相似。 第四章平滑肌細胞重要調節(jié)因子Myocardin的協同轉錄因子的篩選 目的：平滑肌細胞的增殖分化與血管內膜增生,冠狀動脈狹窄等血管病理密切相關,Myocardin是平滑肌分化過程中不可缺少的一個協同轉錄因子,本實驗希望通過篩選與Myocardin結合的蛋白,找到在平滑肌分化過程中新的重要的轉錄因子或協同轉錄因子,研究其在平滑肌分化過程中的功能及所屬信號通路,進而更深入了解平滑肌分化的分子機制。 方法：用熒光素酶活性實驗篩選1170個轉錄因子中能上調Myocardin表達的轉錄因子,并用免疫共沉淀等方法進一步確認候選因子與Myocardin蛋白水平的物理結合,通過生物信息學分析等方法篩選出候選因子,在小鼠胚胎干細胞向平滑肌細胞分化模型中確定其在平滑肌細胞分化中的調節(jié)作用。 結果：我們從1170個轉錄因子中篩選出ZNF297B,RAI14和MAGED1等轉錄因子,確定其在平滑肌中高表達,在平滑肌分化過程中上調,并能與Myocardin直接結合,其過表達能促進平滑肌分化基因的表達。 結論：ZNF297B,RAI14和MAGED1等轉錄因子可能是調節(jié)平滑肌分化的重要轉錄因子。 第五章鋅指蛋白297B協同Myocardin調節(jié)平滑肌細胞的分化 目的：探討鋅指蛋白297B(ZNF297B)在平滑肌細胞分化過程中的表達及相關機制。 方法：用定量實時PCR檢測ZNF297B在人,大鼠,小鼠平滑肌細胞中體內／體外的表達量及在平滑肌細胞體內和體外分化或增殖過程中的表達變化；構建ZNF297B-Myc表達質粒,免疫熒光檢測ZNF297B-Myc在大鼠平滑肌細胞中的表達分布；用熒光素酶活性檢測及免疫共沉淀實驗檢測ZNF297B與Myocardin的結合及相互作用。 結果：ZNF297B在平滑肌細胞中特異性高表達,在小鼠胚胎干細胞向平滑肌細胞分化過程中表達上調,在PDGFBB刺激的平滑肌細胞增殖過程中下調,在大鼠頸動脈球囊損傷內膜修復過程中表達先上調后下降。ZNF297B主要分布于細胞核及核周的細胞質。ZNF297B能與Myocardin蛋白結合,熒光素酶活性檢測顯示ZNF297B的表達受Myocardin的調控。 結論：ZNF297B可能在平滑肌細胞分化過程中起到重要正向調控作用,這一作用很可能是通過ZNF297B與Myocardin的協同作用來完成。
[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.

【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R329

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