炎症细胞因子作用下IGFBP5及KDM6A对牙周膜干细胞成骨分化的作用研究

发布时间：2018-03-21 23:18

本文选题：牙周膜干细胞　切入点：肿瘤坏死因子-α　出处：《天津医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:本研究拟采用丧失性功能实验探讨胰岛素样生长因子结合蛋白5(IGFBP5)和组蛋白去甲基化酶6A(KDM6A)在炎症微环境下对牙周膜干细胞(PDLSCs)成骨分化的影响及分子调控机制,为炎症微环境下牙周组织再生寻找治疗靶点。方法:1.PDLSCs的收集与分离培养,并通过流式细胞术检测其表面抗原标志物。2.分别建立IGFBP5稳定敲低的细胞系,KDM6A稳定敲低的细胞系,并进行成骨诱导分化。在成骨诱导条件培养下过通过CCK-8检测细胞增殖能力,Annexin V检测凋亡变化,碱性磷酸酶活性、碱性磷酸酶染色检测、茜素红染色检测TNF-α、IL-17对牙周膜干细胞分化及生物学特性的影响。3.通过Real-Time PCR检测IGFBP5丧失性功能实验中的PDLSCs在体外成骨诱导条件培养下,炎症因子对PDLSCs的成骨分化相关标志性基因及相关的组蛋白去甲基化酶表达的影响。检测KDM6A低表达后PDLSCs的成骨分化基因表达变化,并通过流式细胞仪微球捕获芯片技术分析其相关炎症因子的变化。4.通过Western-blot检测炎症因子作用下成骨诱导条件培养下KDM6A低表达的PDLSCs,细胞质内p-p65、β-catenin蛋白及细胞核内p-p65蛋白的表达变化。结果:1.通过成骨诱导分化培养基定向诱导IGFBP5低表达的PDLSCs,在炎症因子作用下,碱性磷酸酶染色检测显示蓝染程度减弱。碱性磷酸酶活性检测发现TNF-α、IL-17刺激后的IGFBP5低表达的PDLSCs碱性磷酸酶活性降低。实时定量Real-time RT-PCR结果显示成骨诱导分化组中,TNF-α、IL-17处理后成骨分化的相关基因OCN及BSP表达显著降低,组蛋白去甲基化酶KDM2A、KDM3B、KDM5B、JMJD6也有显著变化。(P0.05,n=3)2.磷酸酶染色结果显示KDM6A低表达的PDLSCs在TNF-α、IL-17刺激后染色效果减弱;茜素红染色结果显示炎症因子刺激下KDM6A低表达后染色效果并无显著变化;通过细胞增殖检测结果可以确定KDM6A丧失性功能实验中的PDLSCs在成骨诱导分化过程中加入TNF-α、IL-17刺激后,与对照组相比并无明显差异。通过细胞凋亡检测结果可以确定KDM6A低表达后的PDLSCs,加入TNF-α、IL-17刺激后细胞凋亡略有增加。Real-Time PCR结果显示在TNF-α、IL-17刺激后,成骨分化重要标志基因RUNX2、BSP、OCN m RNA的表达显著下降。流式细胞仪微球捕获芯片技术检测结果显示KDM6A低表达后的PDLSCs,炎症因子IL-6的表达显著升高。(P0.05,n=3)3.Western-blot检测结果显示,在KDM6A敲低的PDLSCs成骨诱导分化条件培养下,加入TNF-α、IL-17刺激后,细胞质中β-catenin蛋白水平显著降低。结论:1.IGFBP5可以促进炎症因子TNF-α、IL-17刺激下的PDLSCs成骨分化。2.KDM6A可以促进炎症因子TNF-α、IL-17刺激下的PDLSCs成骨分化。3.KDM6A对炎症因子TNF-α、IL-17刺激下PDLSCs成骨分化的促进作用,可能与其促进Wnt/β-catenin信号通路有关。
[Abstract]:Objective: to investigate the effects of insulin-like growth factor-binding protein (IGFBP5) and histone demethylase (6A- KDM6A) on the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in inflammatory microenvironment. Methods: 1. The collection, isolation and culture of PDLSCs were used to detect the surface antigen markers of PDLSCs by flow cytometry. The KDM6A stable knockout cell lines were established by flow cytometry. Under the condition of osteogenic induction, apoptosis, alkaline phosphatase activity and alkaline phosphatase staining were detected by CCK-8 and Annexin V, respectively. Effects of TNF- 伪 IL-17 on differentiation and biological characteristics of periodontal ligament stem cells by alizarin red staining. 3. PDLSCs in IGFBP5 loss function experiment was detected by Real-Time PCR under osteogenic induction condition in vitro. Effects of inflammatory factors on the expression of osteogenic differentiation related signature genes and histone demethylase in PDLSCs. The expression of osteogenic differentiation genes in PDLSCs was detected after low expression of KDM6A. Flow cytometry microsphere capture chip technique was used to analyze the changes of inflammatory cytokines. 4. Western-blot was used to detect PDLSCs, p-p65, 尾 -catenin protein in cytoplasm and p-p65 eggs in nucleus of osteoblasts induced by inflammatory factors. Results 1. The low expression of IGFBP5 was induced by osteogenic differentiation medium. Alkaline phosphatase staining showed that the degree of blue staining was weakened. Alkaline phosphatase activity test showed that the low expression PDLSCs alkaline phosphatase activity of IGFBP5 stimulated by TNF- 伪 IL-17 was decreased. Real-time quantitative Real-time RT-PCR results showed that TNF- 伪 -IL-17 in osteogenic differentiation group was located in TNF- 伪 IL-17. The expression of OCN and BSP in osteogenic differentiation was significantly decreased. The histone demethylase KDM2An KDM3Bm5BHJMJD6 was also significantly changed. The results of phosphatase staining showed that the PDLSCs with low expression of KDM6A decreased after stimulation of TNF- 伪 -IL-17, while the staining effect of alizarin red showed no significant change after the low expression of KDM6A stimulated by inflammatory factors. The results of cell proliferation test showed that PDLSCs in KDM6A loss function test was stimulated by TNF- 伪 -IL-17 during osteogenic induction. Compared with the control group, there was no significant difference between the two groups. The results of apoptosis detection could determine the PDLSCsafter low expression of KDM6A, and increase the apoptosis slightly after adding TNF- 伪 -IL-17. The results of Real-Time PCR showed that the cells were stimulated by TNF- 伪 -IL-17. The expression of OCN m RNA, an important marker of osteogenic differentiation, was significantly decreased. The results of flow cytometry microsphere capture chip showed that the expression of inflammatory factor IL-6 was significantly increased after low expression of KDM6A. Under the condition of KDM6A knockout PDLSCs osteogenesis induced differentiation, TNF- 伪 -IL-17 was added to stimulate the differentiation. Conclusion: 1. IGFBP5 can promote the osteogenic differentiation of PDLSCs stimulated by TNF- 伪 and IL-17. 2. KDM6A can promote the osteogenic differentiation of PDLSCs stimulated by TNF- 伪 -IL-17. 3. KDM6A can promote the osteogenic differentiation of PDLSCs stimulated by TNF- 伪 IL-17. It may be related to the promotion of Wnt/ 尾-catenin signaling pathway.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.4

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