發(fā)布時間：2018-04-20 15:55

  本文選題：表皮生長因子受體 + 毛囊　； 參考：《吉林大學(xué)》2016年博士論文

【摘要】：毛發(fā)的真皮乳頭和真皮鞘中存在著間充質(zhì)干細(xì)胞,它不僅與上皮細(xì)胞相互作用決定了毛發(fā)周期的自我更新,而且也能為疾病的干細(xì)胞移植治療和組織工程器官的構(gòu)建提供自體或異體多能干細(xì)胞來源。臨床研究中發(fā)現(xiàn):間充質(zhì)干細(xì)胞移植治療組織損傷時,單次移植細(xì)胞數(shù)量高達(dá)到1×109個才能有效發(fā)揮治療作用,而短時間內(nèi)可獲得的細(xì)胞數(shù)量不足是阻礙其臨床應(yīng)用的瓶頸。因此如何高效擴增具有自我更新能力和多向分化潛能的間充質(zhì)干細(xì)胞,成為干細(xì)胞再生醫(yī)學(xué)研究和應(yīng)用的基礎(chǔ)和關(guān)鍵。生長因子是一類可以刺激細(xì)胞生長活性的蛋白類成分,參與細(xì)胞增殖、分化和遷移等一系列生物學(xué)過程。成纖維細(xì)胞生長因子(FGF)、表皮生長因子(EGF)、血小板衍生因子(PDGF)、血管內(nèi)皮生長因子(VEGF)均有促進(jìn)細(xì)胞增殖的作用。但生長因子應(yīng)用于人毛囊間充質(zhì)干細(xì)胞的體外擴增,仍有待解決的問題:需要選擇合適的生長因子種類和劑量,使其在促進(jìn)人毛囊間充質(zhì)干細(xì)胞增殖的同時保持其干細(xì)胞的特性,不向分化和癌變的結(jié)局發(fā)展。此外,生長因子促進(jìn)人毛囊間充質(zhì)干細(xì)胞增殖的信號通路還不明確。表皮生長因子(EGF)是一種由53個氨基酸殘基組成的肽類物質(zhì),它與表皮生長因子受體(EGFR)結(jié)合后形成同源二聚體,通過胞內(nèi)激酶區(qū)自身磷酸化,激活下游信號通路,將關(guān)鍵信號分子從細(xì)胞膜傳導(dǎo)到細(xì)胞核內(nèi),啟動相關(guān)基因表達(dá),調(diào)控細(xì)胞的生物學(xué)行為。目前發(fā)現(xiàn)EGFR下游與細(xì)胞增殖相關(guān)的信號通路主要有:Ras-Raf-MAPK-ERK、PI3K-AKT和JAK/STAT信號通路。這些信號通路具有細(xì)胞特異性:不同的細(xì)胞可以通過上述不同的信號通路發(fā)揮生物學(xué)作用。本課題組之前的研究發(fā)現(xiàn):表皮細(xì)胞生長因子(EGF)在人保持毛囊間充質(zhì)干細(xì)胞多潛能分化特性的同時能顯著刺激其增殖。但EGF通過哪條信號通路,促進(jìn)人毛囊間充質(zhì)干細(xì)胞增殖目前尚不清楚。因此,本研究的目的是闡明EGF促進(jìn)人毛囊間充質(zhì)干細(xì)胞增殖的下游信號通路,并初步探討與之先關(guān)的細(xì)胞周期G1-S期的調(diào)控因素。我們用拔取毛發(fā)的方法,從人的毛囊中獲取間充質(zhì)干細(xì)胞,用免疫熒光染色和流式細(xì)胞術(shù)對其進(jìn)行了表面標(biāo)記的鑒定,用多向誘導(dǎo)分化實驗鑒定其多潛能性。用EGF和EGFR抑制劑(AG1478)、PI3K-AKT抑制劑(LY294002)、ERK抑制劑(U0126)以及STAT3抑制劑(STA-21)處理細(xì)胞,檢測EGF以及這幾種抑制劑對人毛囊間充質(zhì)干細(xì)胞增殖的影響和對EGFR及其下游信號通路的激活情況。用western blotting檢測人毛囊間充質(zhì)干細(xì)胞EGF及其下游信號通路對細(xì)胞周期G1-S期調(diào)節(jié)相關(guān)蛋白表達(dá)的影響。研究結(jié)果顯示:我們從毛囊中分離得到的細(xì)胞表達(dá)間充質(zhì)干細(xì)胞的標(biāo)記物:CD90、CD105、CD44、CD73,不表達(dá)CD31。這些細(xì)胞也具有向骨細(xì)胞、脂肪細(xì)胞和軟骨細(xì)胞分化的能力,因此是毛囊間充質(zhì)干細(xì)胞。免疫熒光染色和流式細(xì)胞術(shù)結(jié)果也顯示毛囊間充質(zhì)干細(xì)胞表達(dá)EGFR,并且表達(dá)率在98%以上。細(xì)胞增殖實驗證實1-50 ng/m L EGF可以顯著提高毛囊間充質(zhì)干細(xì)胞的增殖能力,但沒有劑量效應(yīng)關(guān)系。EGFR抑制劑(AG1478,0.2-5μM),PI3K-AKT抑制劑(LY294002,10-50μM),ERK抑制劑(U0126,2-50μM)和STAT3抑制劑(STA-21,20-50μM)顯著抑制EGF引起的毛囊間充質(zhì)干細(xì)胞增殖。Western blotting結(jié)果顯示加入EGF可以上調(diào)p-EGFR、p-ERK和p-AKT蛋白的表達(dá),對p-STAT3沒有作用。而且AG1478-2μM,LY294002-10μM,U0126-10μM分別抑制p-EGFR,p-AKT和p-ERK1/2的表達(dá)。細(xì)胞周期相關(guān)實驗證實EGF(10ng/m L)可以促進(jìn)毛囊間充質(zhì)干細(xì)胞從G1期向S期轉(zhuǎn)變,同時上調(diào)細(xì)胞周期蛋白cyclin D1的表達(dá),下調(diào)p16蛋白表達(dá)。綜上所述,我們的結(jié)果證實EGF通過ERK、AKT信號通路,促進(jìn)人毛囊間充質(zhì)干細(xì)胞增殖。EGF促進(jìn)人毛囊間充質(zhì)干細(xì)胞的細(xì)胞周期從G1期向S期轉(zhuǎn)變,與cyclin D1表達(dá)上調(diào)和p16表達(dá)下調(diào)有關(guān)。這一發(fā)現(xiàn)為毛囊間充質(zhì)干細(xì)胞體外大量擴增提供了新的方法和理論依據(jù),也為毛囊發(fā)育和毛發(fā)周期的研究奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R33

【相似文獻(xiàn)】

相關(guān)期刊論文 前3條

1 袁文佶;黃穎芝;高瓔;石東燕;宗晨;劉立躍;王金福;;人胎盤間充質(zhì)樣干細(xì)胞的分離、培養(yǎng)及其生物學(xué)特性分析[J];浙江大學(xué)學(xué)報(理學(xué)版);2011年03期

2 楊柳;孫海英;胡詩宇;齊念民;;β-TCP三維支架用于骨髓間充質(zhì)干/基質(zhì)細(xì)胞的研究最新進(jìn)展[J];生物技術(shù)通報;2008年S1期

3 ;[J];;年期

相關(guān)會議論文 前10條

1 李杰平;孔佩艷;朱麗丹;孔祥敬;李佳麗;曾東風(fēng);劉紅;王慶余;彭賢貴;陳幸華;張曦;;臍帶間充質(zhì)千細(xì)胞治療15例異基因干細(xì)胞移植術(shù)后慢性GVHD臨床療效觀察[A];第13屆全國實驗血液學(xué)會議論文摘要[C];2011年

2 李秀森;李紅;郭子寬;毛寧;;一種從骨實質(zhì)中分離間充質(zhì)干/祖細(xì)胞的方法[A];第10屆全國實驗血液學(xué)會議論文摘要匯編[C];2005年

3 陳丹丹;陳芳;馬鳳霞;韓忠朝;;Toll樣受體4及其配體脂多糖對臍帶間充質(zhì)干細(xì)胞增殖和分化的影響[A];第13屆全國實驗血液學(xué)會議論文摘要[C];2011年

4 吳永華;;胎兒胰腺來源的單克隆SP細(xì)胞具有間充質(zhì)的表型特征[A];第五次全國中青年檢驗醫(yī)學(xué)學(xué)術(shù)會議論文匯編[C];2006年

5 江宏兵;田衛(wèi)東;湯煒;劉磊;李曉東;;FGF8誘導(dǎo)顱神經(jīng)嵴向第一鰓弓外胚間充質(zhì)分化的實驗研究[A];第三屆全國口腔頜面部創(chuàng)傷暨修復(fù)重建學(xué)術(shù)研討會論文匯編[C];2003年

6 趙宏偉;陳嘹;TsMunkh-aldar;Mungungerel;阿拉坦高勒;;溶血磷脂酸對人臍帶源間充質(zhì)干細(xì)胞增殖及表面標(biāo)記物表達(dá)的影響[A];第11屆全國脂質(zhì)與脂蛋白學(xué)術(shù)會議論文匯編[C];2012年

7 曾慧蘭;覃永亮;鐘啟;卜欠欠;韓新愛;;尼古丁對人臍帶間充質(zhì)干細(xì)胞增殖、凋亡及蛋白差異表達(dá)的影響[A];第13屆全國實驗血液學(xué)會議論文摘要[C];2011年

8 李紅;李秀森;毛寧;;間充質(zhì)干細(xì)胞輸注可從其三個階段抑制急性移植物抗宿主病的發(fā)生發(fā)展[A];第12屆全國實驗血液學(xué)會議論文摘要[C];2009年

9 李志堅;;腹部間充質(zhì)腫瘤的CT診斷[A];2006年華東六省一市暨浙江省放射學(xué)學(xué)術(shù)年會論文匯編[C];2006年

10 廖聯(lián)明;郭虹;劉津華;楊少光;王瑛慧;劉杰文;趙春華;;TGF浜1對胎兒骨髓來源的CD105~+間充質(zhì)干細(xì)胞增殖和分化的影響[A];第九屆全國實驗血液學(xué)會議論文摘要匯編[C];2003年

相關(guān)博士學(xué)位論文 前1條

1 白婷婷;表皮生長因子促進(jìn)人毛囊間充質(zhì)干細(xì)胞增殖的機制研究[D];吉林大學(xué);2016年

相關(guān)碩士學(xué)位論文 前3條

1 袁文佶;人胎盤間充質(zhì)樣干細(xì)胞的分離、培養(yǎng)及其生物學(xué)特性分析[D];浙江大學(xué);2011年

2 黃林生;雞胚真皮間充質(zhì)干/祖細(xì)胞分離培養(yǎng)與誘導(dǎo)分化研究[D];湖南農(nóng)業(yè)大學(xué);2011年

3 鐘啟;缺氧對臍帶間充質(zhì)干細(xì)胞增殖及差異蛋白表達(dá)譜的影響[D];暨南大學(xué);2011年

，

本文編號：1778438