Periostin对牙周膜干细胞定向分化为神经细胞作用的影响

发布时间：2018-01-06 19:08

  本文关键词：Periostin对牙周膜干细胞定向分化为神经细胞作用的影响　出处：《南京医科大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： Periostin 牙周膜干细胞 分化 神经细胞

【摘要】：研究背景神经的构建在生理性牙周膜重建的过程中是不可或缺的重要环节,对于维持牙周膜的完整性以及牙周膜功能的行使具有一定意义。因此,牙周膜内神经的重建对于种植体周围生理性牙周膜的构建有重要意义。既往研究发现,牙周膜干细胞有向神经细胞分化的潜能,为生理性牙周膜的重建提供了重要的理论依据。因此,如何有效地促进牙周膜干细胞分化为神经细胞是本课题的核心内容。实验目的拟通过研究Periostin对牙周膜干细胞(hPDLSCs)定向分化为神经细胞的作用及调控机制,进而探索重建生理性牙周膜的可能,为种植体周围生理性牙周膜的构建提供重要理论依据。实验方法第一部分:人牙周膜干细胞的培养及诱导分化1.1人牙周膜干细胞(hPDLSCs)的诱导培养对照组:将hPDLSCs接种于六孔板,加入bFGF预诱导液,诱导液中bFGF的浓度为10ng/mL,在诱导24h后换液,用PBS轻轻洗涤三次后再加入诱导液,继续进行诱导培养,隔天换液并拍照。实验组:加入诱导液同时添加100 ng/mL的Periostin外源性刺激的牙周膜干细胞继续诱导培养,隔天换液并拍照。1.2 Western blot、免疫荧光检测牙周膜干细胞成神经相关蛋白(β-tubulinⅢ、GFAP、NSE)的表达。第二部分:沉默牙周膜干细胞Periostin的表达对hPDLSCs定向分化为神经细胞的影响2.1构建Periostin基因干扰慢病毒,RT-PCR法检测慢病毒LVX-PERIOSTIN-RNAi 下调 PERIOSTIN 基因表达效率2.2 Periostin基因的干扰慢病毒感染hPDLSCs细胞,WB、免疫荧光检测牙周膜干细胞成神经相关蛋白(β-tubulin Ⅲ、GFAP、NSE)的表达。实验结果1.培养的hPDLSCs细胞呈现多角形、不规则或者是梭型。经过Periostin刺激诱导分化后,细胞逐渐变为长梭形,呈现出神经细胞的特征性形态。2.WB及免疫荧光染色显示,在hPDLSCs细胞中,β-tubulin Ⅲ、GFAP、NSE均呈低表达,而经成神经诱导培养后,其β-tubulin Ⅲ、GFAP、NSE表达水平显著升高,在此基础上,使用外源性Periostin刺激后,β-tubulin Ⅲ、GFAP、NSE表达水平进一步升高。WB结果显示,在培养第4天时,β-tubulinⅢ、GFAP、NSE表达水平达到一个相对的高峰,在诱导培养第7天时,β-tubulin Ⅲ、GFAP、NSE表达水平较第4天时有所下降。3.我们成功构建了 LVX-PERIOSTIN-RNAi,并使用其感染hPDLSCs细胞。进一步采用western blot、免疫荧光法检测了 LVX-PERIOSTIN-RNAi感染后神经细胞特异性相关蛋白的表达。结果显示,在感染空载体后,外源性Periostin能够显著上调神经细胞特异性相关蛋白β-tubulin Ⅲ、GFAP、NSE 的表达。而在感染 LVX-PERIOSTIN-RNAi 后,β-tubulin Ⅲ、GFAP、NSE的表达水平较空载体组显著下降。结论1.外源性Periostin能够促进牙周膜干细胞定向分化为神经细胞;2.慢病毒沉默牙周膜干细胞Periostin的表达,能够显著抑制牙周膜干细胞定向分化为神经细胞。
[Abstract]:Background the construction of nerve is an indispensable link in the process of physiological periodontal ligament reconstruction, and it is of certain significance to maintain the integrity of periodontal ligament and the function of periodontal ligament. The reconstruction of periodontal ligament nerve plays an important role in the construction of physiological periodontal ligament around implants. Previous studies have found that periodontal ligament stem cells have the potential to differentiate into neural cells. It provides an important theoretical basis for the reconstruction of physiological periodontal ligament. How to effectively promote the differentiation of periodontal ligament stem cells into nerve cells is the core of this study. The role and regulatory mechanism of directional differentiation into nerve cells. To explore the possibility of reconstruction of physiological periodontal membrane. An important theoretical basis for the construction of physiological periodontal ligament around implants. Part one: culture and differentiation of Human periodontal ligament Stem cells (hPDLSCs). HPDLSCs was inoculated on the six-hole plate. The concentration of bFGF in the induction solution was 10ng / mL by adding bFGF pre-induction solution. After 24 hours of induction, the solution was changed, then gently washed with PBS for three times, and then added to the inducer solution, and then the induction culture was carried out. The experiment group: the periodontal ligament stem cells stimulated by 100 ng/mL Periostin were added to the solution and cultured continuously. The next day we changed the fluid and photographed. 1.2 Western blot.The immunofluorescence was used to detect the expression of 尾 -tubulin 鈪，

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