转录因子CREB在根尖牙乳头干细胞定向分化中的作用

发布时间：2018-04-04 02:26

本文选题：CREB　切入点：根尖牙乳头干细胞　出处：《安徽医科大学》2016年硕士论文

【摘要】：近年来,干细胞生物学和组织工程学发展迅速,这促进了再生性医学的进步。再生性医学的目的是创造功能性、有活力的组织,修复或取代受损的组织。在牙体牙髓科中,根尖牙乳头干细胞(SCAPs,stem cells from the apical papilla)是研究的热点,它存在于未发育完全的恒牙根尖周组织中,是具有高度增生自我更新能力和多向分化潜能的成体干细胞,被认为是牙根部成牙本质细胞的来源,最终形成牙根部牙本质。SCAPs的存在解释了一些被感染的年轻恒牙经过适当的消毒处理后,牙根仍能继续发育的现象。cAMP/PKA/CREB信号通路可以决定间充质干细胞的成骨向分化潜力。作为该通路的下游信号,CREB(c AMP responsive element-binding protein)起了关键作用,它通过附着于c AMP(cyclic Adenosine 3',5'-monophosphate)反应基因的保守序列而发挥作用。已经发现在人类磨牙成牙本质细胞和成牙骨质细胞的细胞核内,CREB表达明显,而抑制CREB后,小鼠的软骨内骨形成减少。此外,在许多与矿化过程相关基因的启动子区域内,含有转录因子CREB在靶基因上的结合位点CRE,如骨钙素(OCN,osteocalcin),骨涎蛋白(BSP,bone sialoprotein)等。提示CREB在生物矿化的过程中起了重要作用,然而,在SCAPs中,CREB对于成牙本质/成骨分化的作用还未完全确定。目的通过慢病毒转染人根尖牙乳头干细胞(SCAPs,stem cells from the apical papilla),改变转录因子CREB(c AMP-responsive element binding protein)在SCAPs中的表达,检测对SCAPs成牙本质/成骨向分化的影响,从而探讨CREB定向调控根尖牙乳头干细胞分化的分子机制。方法分离培养人根尖牙乳头干细胞;采用有限稀释法获得克隆化细胞;流式细胞术和细胞多向分化诱导实验鉴定实验细胞的组织学来源;分别构建慢病毒载体介导的CREB基因过表达和沉默的根尖牙乳头干细胞;实时定量PCR和Western Blot检测慢病毒转染效率。预实验确立各组慢病毒MOI(Multiply of Infection)值及最佳转染条件。将细胞分组为空白对照组、阴性病毒对照组、过表达组、沉默组。将空白对照组、病毒对照组、CREB过表达/沉默组矿化诱导2周后,茜素红染色观察比较矿化结节形成的量,并半定量分析;实时定量PCR检测矿化诱导1、2、3周时,各组细胞的碱性磷酸酶(alkaline phosphateasea,ALP),Ⅰ型胶原(collagen typeⅠ,Col-Ⅰ),runt相关转录因子2(RUNX2),骨钙素(osteocalcin,OCN),osterix(OSX)的m RNA表达;对各组细胞矿化诱导3周后,Western Blot检测成牙本质特异性蛋白DSP(dentin sialoprotein)及成骨分化标志物RUNX2的表达,并进行蛋白半定量分析。结果流式细胞术和干细胞多向分化实验证实分离培养的细胞为SCAPs。实时定量PCR和Western Blot检测显示慢病毒转染SCAPs后能够有效改变目的基因CREB表达,结合荧光显微镜观察说明慢病毒具有很高的转染效率,且对细胞生长无明显影响。茜素红染色及矿化结节半定量分析显示,矿化诱导2周后,CREB过表达组形成钙结节能力增强;矿化诱导1、2、3周,CREB过表达组中的矿化标志物ALP,Col-Ⅰ,RUNX2,OCN,OSX的m RNA在各时间点几乎均上升;Western Blot检测矿化诱导3周时的细胞示,CREB过表达组DSP,RUNX2蛋白表达升高(P0.05)。而CREB沉默组形成钙结节能力则减弱(P0.05),且在矿化诱导4、7天时检测,矿化基因的m RNA均有不同程度的降低(P0.05)。结论在SCAPs矿化诱导过程中,过表达CREB促进SCAPs向成牙/成骨细胞分化,而沉默CREB则发挥相反作用,提示CREB可能促进SCAPs的定向分化能力。为牙源性干细胞在牙髓/牙本质再生和生物性牙根的形成提供新的思路。
[Abstract]:In recent years, stem cell biology and tissue engineering develops rapidly, which promotes the regeneration of the progress of medicine. Medical regeneration is to create functional, vibrant organization, repair or replace damaged tissue. In the Department of endodontics, SCAP (SCAPs, stem cells from the apical papilla) is a hotspot of research, it exists in immature permanent teeth in periapical tissues, is highly proliferative self-renewal and multilineage differentiation potential of adult stem cells, is considered to be the root source of odontoblasts, finally form the root dentin.SCAPs exists to explain some of the infected young permanent teeth after disinfection treatment after appropriate, the phenomenon of.CAMP/PKA/CREB signaling pathway can continue to root development can determine mesenchymal stem cells and osteogenic differentiation potential. As the downstream signal pathway, CREB (C AMP Responsive element-binding protein) played a key role, it attached to the C AMP (cyclic Adenosine 3', 5'-monophosphate) conserved sequence response genes play a role. Have been found in human molar odontoblasts and cementum cell nucleus, the expression of CREB significantly, and the inhibition of CREB mice after endochondral bone formation decreased. In addition, the promoter region in many mineralization related genes, containing CRE binding sites of transcription factor CREB in the target genes, such as Osteocalcin (OCN, osteocalcin), bone sialoprotein (BSP, bone, sialoprotein). CREB plays an important role in the process of biomineralization, however, in SCAPs, CREB for odontogenic / osteogenic differentiation has not been fully established. The purpose of stem cells by lentivirus transfected SCAP (SCAPs, stem cells from the apical papilla), the change of transcription factors CREB (C AMP-responsive element binding protein) expression in SCAPs, detection of SCAPs odontoblast / osteogenic differentiation effect, so as to explore the molecular mechanism of CREB cell differentiation and regulation of directional SCAP. Isolation and culture of human SCAP method; acquired cell cloned by limited dilution method; the source of flow cytometry and differentiation induced by experimental identification of experimental cells respectively; construction of CREB gene lentiviral vector mediated over expression and silencing of the apical papilla stem cells; quantitative real-time PCR and Western detection of Blot lentiviral transfection efficiency. The pre experiment establishment of lentiviral MOI groups (Multiply of Infection) value and the optimal transfection conditions. The cells were divided into control group, negative control virus group, overexpression group, silence will group. The blank control group, virus control group, over expression of CREB / silent group induced mineralization By 2 weeks, alizarin red staining to observe and compare the formation of mineralized nodules, and semi quantitative analysis; real-time quantitative PCR detection of mineralization induced by 1,2,3 weeks, the cells were alkaline phosphatase (alkaline phosphateasea, ALP), collagen type I (collagen type I, Col- I), runt related transcription factor 2 (RUNX2). Osteocalcin (osteocalcin, OCN), osterix (OSX) expression of M RNA; to each cell mineralization after 3 weeks of induction, Western Blot detection of odontoblast specific protein DSP (dentin sialoprotein) and osteogenic differentiation marker RUNX2 expression, and semi quantitative analysis of protein. The results of flow cytometry and multipotential stem cell differentiation experiments confirmed that the cultured cells showed that lentiviral transfection of SCAPs SCAPs. and Western Blot real-time quantitative PCR detection can effectively change the gene expression of CREB, combined with fluorescence microscopy that lentivirus with high transfection efficiency, The growth and has no obvious effect on the cells. Semi quantitative alizarin red staining and mineralized nodules analysis showed that 2 weeks after the induction of CREB mineralization, formation of calcium nodules and enhance the ability of expression; mineralization induced by 1,2,3 weeks, over expression of CREB in the group of mineralization markers ALP, Col- 1, RUNX2, OCN, m RNA OSX in the almost all time points showed rise; at 3 weeks of Western cells induced by Blot detection of mineralization, overexpression of CREB group DSP, RUNX2 protein expression increased (P0.05). CREB silencing group calcium nodules formation ability decreased (P0.05), and the detection in mineralization induced 4,7 days, decrease mineralization gene of M RNA was different the (P0.05). Conclusion in SCAPs induced mineralization process, overexpression of CREB promotes SCAPs into the tooth / osteoblast differentiation, whereas knockdown of CREB will play the opposite effect, suggesting that CREB may promote the differentiation ability of SCAPs. For dental stem cells in dental pulp / dentin regeneration and biological The formation of the root provides a new way of thinking.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R781

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