表皮生长因子促进人毛囊间充质干细胞增殖的机制研究

发布时间：2018-04-20 15:55

本文选题：表皮生长因子受体 + 毛囊　；参考：《吉林大学》2016年博士论文

【摘要】：毛发的真皮乳头和真皮鞘中存在着间充质干细胞,它不仅与上皮细胞相互作用决定了毛发周期的自我更新,而且也能为疾病的干细胞移植治疗和组织工程器官的构建提供自体或异体多能干细胞来源。临床研究中发现:间充质干细胞移植治疗组织损伤时,单次移植细胞数量高达到1×109个才能有效发挥治疗作用,而短时间内可获得的细胞数量不足是阻碍其临床应用的瓶颈。因此如何高效扩增具有自我更新能力和多向分化潜能的间充质干细胞,成为干细胞再生医学研究和应用的基础和关键。生长因子是一类可以刺激细胞生长活性的蛋白类成分,参与细胞增殖、分化和迁移等一系列生物学过程。成纤维细胞生长因子(FGF)、表皮生长因子(EGF)、血小板衍生因子(PDGF)、血管内皮生长因子(VEGF)均有促进细胞增殖的作用。但生长因子应用于人毛囊间充质干细胞的体外扩增,仍有待解决的问题:需要选择合适的生长因子种类和剂量,使其在促进人毛囊间充质干细胞增殖的同时保持其干细胞的特性,不向分化和癌变的结局发展。此外,生长因子促进人毛囊间充质干细胞增殖的信号通路还不明确。表皮生长因子(EGF)是一种由53个氨基酸残基组成的肽类物质,它与表皮生长因子受体(EGFR)结合后形成同源二聚体,通过胞内激酶区自身磷酸化,激活下游信号通路,将关键信号分子从细胞膜传导到细胞核内,启动相关基因表达,调控细胞的生物学行为。目前发现EGFR下游与细胞增殖相关的信号通路主要有:Ras-Raf-MAPK-ERK、PI3K-AKT和JAK/STAT信号通路。这些信号通路具有细胞特异性:不同的细胞可以通过上述不同的信号通路发挥生物学作用。本课题组之前的研究发现:表皮细胞生长因子(EGF)在人保持毛囊间充质干细胞多潜能分化特性的同时能显著刺激其增殖。但EGF通过哪条信号通路,促进人毛囊间充质干细胞增殖目前尚不清楚。因此,本研究的目的是阐明EGF促进人毛囊间充质干细胞增殖的下游信号通路,并初步探讨与之先关的细胞周期G1-S期的调控因素。我们用拔取毛发的方法,从人的毛囊中获取间充质干细胞,用免疫荧光染色和流式细胞术对其进行了表面标记的鉴定,用多向诱导分化实验鉴定其多潜能性。用EGF和EGFR抑制剂(AG1478)、PI3K-AKT抑制剂(LY294002)、ERK抑制剂(U0126)以及STAT3抑制剂(STA-21)处理细胞,检测EGF以及这几种抑制剂对人毛囊间充质干细胞增殖的影响和对EGFR及其下游信号通路的激活情况。用western blotting检测人毛囊间充质干细胞EGF及其下游信号通路对细胞周期G1-S期调节相关蛋白表达的影响。研究结果显示:我们从毛囊中分离得到的细胞表达间充质干细胞的标记物:CD90、CD105、CD44、CD73,不表达CD31。这些细胞也具有向骨细胞、脂肪细胞和软骨细胞分化的能力,因此是毛囊间充质干细胞。免疫荧光染色和流式细胞术结果也显示毛囊间充质干细胞表达EGFR,并且表达率在98%以上。细胞增殖实验证实1-50 ng/m L EGF可以显著提高毛囊间充质干细胞的增殖能力,但没有剂量效应关系。EGFR抑制剂(AG1478,0.2-5μM),PI3K-AKT抑制剂(LY294002,10-50μM),ERK抑制剂(U0126,2-50μM)和STAT3抑制剂(STA-21,20-50μM)显著抑制EGF引起的毛囊间充质干细胞增殖。Western blotting结果显示加入EGF可以上调p-EGFR、p-ERK和p-AKT蛋白的表达,对p-STAT3没有作用。而且AG1478-2μM,LY294002-10μM,U0126-10μM分别抑制p-EGFR,p-AKT和p-ERK1/2的表达。细胞周期相关实验证实EGF(10ng/m L)可以促进毛囊间充质干细胞从G1期向S期转变,同时上调细胞周期蛋白cyclin D1的表达,下调p16蛋白表达。综上所述,我们的结果证实EGF通过ERK、AKT信号通路,促进人毛囊间充质干细胞增殖。EGF促进人毛囊间充质干细胞的细胞周期从G1期向S期转变,与cyclin D1表达上调和p16表达下调有关。这一发现为毛囊间充质干细胞体外大量扩增提供了新的方法和理论依据,也为毛囊发育和毛发周期的研究奠定了基础。
[Abstract]:Mesenchymal stem cells exist in the dermal papilla and the dermal sheath of hair, which not only interact with epithelial cells to determine the self renewal of the hair cycle, but also provide autologous or allogenic pluripotent stem cells for stem cell transplantation and construction of tissue engineering organs. In the treatment of tissue injury, the number of single transplanted cells is up to 1 * 109 to play the role of treatment effectively, and the shortage of cells in a short period of time is the bottleneck that hinders its clinical application. Therefore, how to efficiently amplify the mesenchymal stem cells with the ability of self renewal and multidirectional differentiation to become the research of stem cell regeneration medicine The growth factor is a class of proteins that can stimulate cell growth activity, participate in a series of biological processes, such as cell proliferation, differentiation and migration, such as fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet derived factor (PDGF) and vascular endothelial growth factor (VEGF). But growth factors are applied to the expansion of human hair follicle mesenchymal stem cells in vitro. The problem remains to be solved: the need to select suitable growth factors and doses to promote the proliferation of human hair follicle mesenchymal stem cells and to maintain the characteristics of their stem cells, not to the outcome of differentiation and cancer. In addition, growth factors promote the growth factor. The signal pathway for the proliferation of human hair follicle mesenchymal stem cells is not clear. Epidermal growth factor (EGF) is a peptide substance composed of 53 amino acid residues. It combines with epidermal growth factor receptor (EGFR) to form homologous two polymer and activates downstream signaling pathway through the phosphorylation of the intracellular kinase area, and the key signal molecules are from thin. The cell membrane conduction into the nucleus, starting the related gene expression and regulating the biological behavior of the cell. It is found that the signal pathways related to cell proliferation in the downstream of EGFR are mainly: Ras-Raf-MAPK-ERK, PI3K-AKT and JAK/STAT signaling pathways. These signaling pathways are cell specific: different cells can be transmitted through the different signaling pathways above. Previous studies in this group have found that epidermal growth factor (EGF) can stimulate the multipotential differentiation of human hair follicle mesenchymal stem cells at the same time and stimulate its proliferation. But which signal pathway of EGF is not clear before the proliferation of human hair follicle mesenchymal stem cells is not clear. Therefore, the purpose of this study is to clarify EGF The downstream signal pathway to promote the proliferation of human hair follicle mesenchymal stem cells and the preliminary discussion of the regulatory factors of the G1-S phase of the cell cycle that are first closed. We use the method of extraction of hair to obtain mesenchymal stem cells from the human hair follicle, and use immunofluorescence staining and flow cytometry to identify the surface markers, and use the multi direction differentiation to induce differentiation. Experiments were conducted to identify the multipotency. Cells were treated with EGF and EGFR inhibitors (AG1478), PI3K-AKT inhibitors (LY294002), ERK inhibitors (U0126) and STAT3 inhibitors (STA-21). The effects of EGF and these inhibitors on the proliferation of human hair follicle mesenchymal stem cells and the activation of EGFR and downstream signal pathways were detected by western. The effect of human hair follicle mesenchymal stem cells EGF and its downstream signal pathway on the expression of regulation related protein in cell cycle G1-S. The results showed that the cells isolated from the hair follicles expressed markers of mesenchymal stem cells: CD90, CD105, CD44, CD73, and non CD31. cells also had the osteoblasts, adipocytes and cartilage. The ability of cell differentiation was the hair follicle mesenchymal stem cells. The results of immunofluorescence staining and flow cytometry also showed that the follicle mesenchymal stem cells expressed EGFR, and the expression rate was over 98%. Cell proliferation experiments confirmed that 1-50 ng/m L EGF could significantly increase the proliferation ability of the hair follicle mesenchymal stem cells, but there was no dose effect relationship.EGFR. The inhibitor (AG1478,0.2-5 mu M), the PI3K-AKT inhibitor (LY294002,10-50 mu M), the ERK inhibitor (U0126,2-50 mu M) and the STAT3 inhibitor (STA-21,20-50 mu M) significantly inhibit the proliferation of the follicle mesenchymal stem cells. 8-2 mu M, LY294002-10 M, and U0126-10 mu M inhibit the expression of p-EGFR, p-AKT and p-ERK1/2 respectively. Cell cycle related experiments confirm that EGF (10ng/m L) can promote the transformation of mesenchymal stem cells from the G1 phase to the phase of the G1 phase, and up regulate the expression of cyclin protein and down regulate the expression of white egg white. The T signaling pathway, promoting the proliferation of human hair follicle mesenchymal stem cells (.EGF), promotes the cell cycle of human hair follicle mesenchymal stem cells from G1 phase to S phase, which is related to the up regulation of cyclin D1 expression and the downregulation of p16 expression. This discovery provides a new formula and theoretical basis for the large expansion of follicle mesenchymal stem cells in vitro, as well as hair follicle development and hair. The study of the cycle has laid the foundation.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R33

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