【摘要】：微環(huán)境可以影響干細(xì)胞的功能,炎癥微環(huán)境下牙髓干細(xì)胞的功能也受到一定的影響。而關(guān)于炎癥微環(huán)境下DPSCs的生物學(xué)行為改變的報(bào)道結(jié)果不一,其具體分子機(jī)制尚不清楚。本文將分三個(gè)部分就這一問題進(jìn)行探討分析： 第一部分炎癥微環(huán)境下牙髓干細(xì)胞的生物學(xué)行為分析 目的：通過比較正常牙髓干細(xì)胞與炎癥牙髓干細(xì)胞的增殖能力和炎性因子的表達(dá)情況分析炎癥微環(huán)境對DPSCs生物學(xué)行為的影響。方法：1)采用有限稀釋法從正常及炎癥牙髓組織中分離培養(yǎng)得到n DPSCs與i DPSCs。2) Western Blot、 real time RT-PCR檢測兩種細(xì)胞的炎性因子IL-1β和TNF-α表達(dá)情況。3)流式細(xì)胞儀及免疫熒光染色檢測間充質(zhì)干細(xì)胞表面標(biāo)記CD45, CD34, CD90, CD105, CD146。4)通過繪制生長曲線、克隆形成率,比較兩種細(xì)胞的增殖能力。結(jié)果：炎癥牙髓組織中牙髓干細(xì)胞的炎癥因子表達(dá)高于正常牙髓干細(xì)胞(P0.05)。流式結(jié)果顯示,炎癥牙髓干細(xì)胞CD45, CD34陰性,CD90, CD105, CD146陽性,免疫熒光顯示VM染色陽性,CK陰性,是間充質(zhì)來源的干細(xì)胞。MTT結(jié)果顯示炎癥牙髓干細(xì)胞的增殖活性高于正常牙髓干細(xì)胞(P0.05)。結(jié)論：本研究成功分離培養(yǎng)獲得了炎癥組織來源的牙髓干細(xì)胞,較正常牙髓干細(xì)胞具有更強(qiáng)的增殖能力。 第二部分炎癥微環(huán)境下牙髓干細(xì)胞成骨／成牙能力的研究 目的：研究炎癥微環(huán)境下牙髓干細(xì)胞的成骨／成牙能力。方法：采用ALP活性檢測、Western Blot及real time RT-PCR檢測成骨/牙相關(guān)蛋白及基因(RUNX2, DSP,DMP1,OSX,OPN,BSP及OCN)的表達(dá)水平。結(jié)果：在蛋白和基因水平,第3代炎癥牙髓干細(xì)胞RUNX2,DSP,DNP1,OSX,OPN,BSP及OCN的表達(dá)均較正常牙髓干細(xì)胞高,但是這種差異隨著傳代次數(shù)的增加而變得不明顯。結(jié)論：炎癥牙髓干細(xì)胞具有更強(qiáng)的成骨／成牙能力。 第三部分Wnt信號通路在炎癥牙髓干細(xì)胞成骨/成牙分化中的作用研究 目的：探討Wnt信號通路在炎癥微環(huán)境下DPSCs成骨／成牙分化過程中的作用及機(jī)制。方法：1)成骨誘導(dǎo)7d后,Western blot檢測Wnt信號通路相關(guān)蛋白GSK3β, β-catenin,CaMKⅡ與NLK的表達(dá)。2)LiCl,DKK-1作用于兩種細(xì)胞后,檢測Wnt信號通路相關(guān)蛋白的表達(dá),同時(shí)檢測成骨相關(guān)基因RUNX2與OCN的表達(dá)。結(jié)果：1)成骨誘導(dǎo)7d后,Western blot結(jié)果顯示,β-catenin與NLK的表達(dá)較正常牙髓干細(xì)胞上調(diào),GSK3β的表達(dá)較正常牙髓干細(xì)胞表達(dá)降低,CaMKⅡ的表達(dá)未見明顯差異。2)炎癥牙髓干細(xì)胞中,LiCl激活Wnt信號通路后β-catenin及LEF-1的表達(dá)水平升高,RUNX2,OCN與DSPP的表達(dá)上調(diào)；DKK-1阻斷Wnt信號通路之后β-catenin,CaMKⅡ,NLK表達(dá)水平下降,RUNX2,OCN與DSPP的表達(dá)下調(diào)。結(jié)論：炎癥微環(huán)境下牙髓干細(xì)胞Wnt信號通路被激活,牙髓干細(xì)胞骨向分化能力增強(qiáng)。
[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- 偽 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- 偽 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，

本文編號：2145568