炎症微环境下牙髓干细胞生物学行为改变的机制研究

发布时间：2018-07-26 09:23

【摘要】：微环境可以影响干细胞的功能,炎症微环境下牙髓干细胞的功能也受到一定的影响。而关于炎症微环境下DPSCs的生物学行为改变的报道结果不一,其具体分子机制尚不清楚。本文将分三个部分就这一问题进行探讨分析： 第一部分炎症微环境下牙髓干细胞的生物学行为分析 目的：通过比较正常牙髓干细胞与炎症牙髓干细胞的增殖能力和炎性因子的表达情况分析炎症微环境对DPSCs生物学行为的影响。方法：1)采用有限稀释法从正常及炎症牙髓组织中分离培养得到n DPSCs与i DPSCs。2) Western Blot、 real time RT-PCR检测两种细胞的炎性因子IL-1β和TNF-α表达情况。3)流式细胞仪及免疫荧光染色检测间充质干细胞表面标记CD45, CD34, CD90, CD105, CD146。4)通过绘制生长曲线、克隆形成率,比较两种细胞的增殖能力。结果：炎症牙髓组织中牙髓干细胞的炎症因子表达高于正常牙髓干细胞(P0.05)。流式结果显示,炎症牙髓干细胞CD45, CD34阴性,CD90, CD105, CD146阳性,免疫荧光显示VM染色阳性,CK阴性,是间充质来源的干细胞。MTT结果显示炎症牙髓干细胞的增殖活性高于正常牙髓干细胞(P0.05)。结论：本研究成功分离培养获得了炎症组织来源的牙髓干细胞,较正常牙髓干细胞具有更强的增殖能力。 第二部分炎症微环境下牙髓干细胞成骨／成牙能力的研究 目的：研究炎症微环境下牙髓干细胞的成骨／成牙能力。方法：采用ALP活性检测、Western Blot及real time RT-PCR检测成骨/牙相关蛋白及基因(RUNX2, DSP,DMP1,OSX,OPN,BSP及OCN)的表达水平。结果：在蛋白和基因水平,第3代炎症牙髓干细胞RUNX2,DSP,DNP1,OSX,OPN,BSP及OCN的表达均较正常牙髓干细胞高,但是这种差异随着传代次数的增加而变得不明显。结论：炎症牙髓干细胞具有更强的成骨／成牙能力。 第三部分Wnt信号通路在炎症牙髓干细胞成骨/成牙分化中的作用研究 目的：探讨Wnt信号通路在炎症微环境下DPSCs成骨／成牙分化过程中的作用及机制。方法：1)成骨诱导7d后,Western blot检测Wnt信号通路相关蛋白GSK3β, β-catenin,CaMKⅡ与NLK的表达。2)LiCl,DKK-1作用于两种细胞后,检测Wnt信号通路相关蛋白的表达,同时检测成骨相关基因RUNX2与OCN的表达。结果：1)成骨诱导7d后,Western blot结果显示,β-catenin与NLK的表达较正常牙髓干细胞上调,GSK3β的表达较正常牙髓干细胞表达降低,CaMKⅡ的表达未见明显差异。2)炎症牙髓干细胞中,LiCl激活Wnt信号通路后β-catenin及LEF-1的表达水平升高,RUNX2,OCN与DSPP的表达上调；DKK-1阻断Wnt信号通路之后β-catenin,CaMKⅡ,NLK表达水平下降,RUNX2,OCN与DSPP的表达下调。结论：炎症微环境下牙髓干细胞Wnt信号通路被激活,牙髓干细胞骨向分化能力增强。
[Abstract]:Microenvironment can affect the function of stem cells, and the function of dental pulp stem cells in inflammatory microenvironment is also affected. However, the results of biological behavior changes of DPSCs in inflammatory microenvironment are different, and its molecular mechanism is not clear. This paper will discuss and analyze this problem in three parts: part one: biological behavior analysis of dental pulp stem cells in inflammatory microenvironment objective: to compare normal dental pulp stem cells with inflammation The proliferative ability of dental pulp stem cells and the expression of inflammatory factors were analyzed. The effects of inflammatory microenvironment on the biological behavior of DPSCs were analyzed. Methods: the expression of inflammatory cytokines IL-1 尾 and TNF- 伪 in normal and inflammatory dental pulp tissues were detected by DPSCs and I DPSCs.2) Western Blot, real time RT-PCR by using the limited dilution method. 3) flow cytometry and immunofluorescence staining were used to detect the expression of IL-1 尾 and TNF- 伪 in the two kinds of cells. The surface markers CD45, CD34, CD90, CD105, CD146.4) of mesenchymal stem cells were drawn by drawing the growth curve. Clone formation rate, compare the proliferative ability of two kinds of cells. Results: the expression of inflammatory factors in dental pulp stem cells was higher than that in normal dental pulp stem cells (P0.05). The results of flow cytometry showed that CD45, CD34 negative CD90, CD105, CD146 were positive, immunofluorescence showed VM staining positive and CK negative. The results showed that the proliferation activity of inflammatory dental pulp stem cells was higher than that of normal dental pulp stem cells (P0.05). Conclusion: dental pulp stem cells derived from inflammatory tissue were successfully isolated and cultured in this study, which have stronger proliferative ability than normal dental pulp stem cells. The second part of the study on osteogenesis / tooth formation ability of dental pulp stem cells in inflammatory microenvironment objective: to study the osteogenesis / tooth formation ability of dental pulp stem cells in inflammatory microenvironment. Methods: the expression of osteoblast / dental associated protein (RUNX2, DSPDMP1, OSXOP-OPN, BSP and OCN) was detected by ALP activity assay by Western Blot and real time RT-PCR. Results: at the level of protein and gene, the expression of BSP and OCN in the third generation of inflammatory dental pulp stem cells (RUNX2DSP1) was higher than that in normal dental pulp stem cells, but the difference was not obvious with the increase of passage times. Conclusion: inflammatory dental pulp stem cells have stronger osteogenic / dental ability. Part three the role of Wnt signaling pathway in osteogenesis / odontogenesis of inflammatory dental pulp stem cells objective: to investigate the role and mechanism of Wnt signaling pathway in the process of DPSCs osteogenesis / tooth differentiation in inflammatory microenvironment. Methods 7 days after osteogenesis induction, the expression of GSK3 尾, 尾 -cateninine CaMK 鈪，

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