【作者】 王颖；

【导师】 李锐；

【作者基本信息】 郑州大学 ， 口腔临床医学， 2020， 硕士

【摘要】 目的细胞外微环境可影响间充质干细胞的分化能力,尤其是炎症微环境。以往的研究表明,炎症刺激下的间充质干细胞可高表达肿瘤坏死因子诱导蛋白6(TSG-6),这种蛋白可以抑制骨髓间充质干细胞的矿化。高浓度的肿瘤坏死因子(TNF-α)可抑制牙髓干细胞(DPSCs)的成牙/成骨分化,然而,TNF-α对DPSCs成牙/成骨能力的抑制是否与TSG-6有关,尚需进一步研究。本研究以增强牙髓干细胞在炎症环境中分化能力的因素为切入点,为牙髓炎症的治疗寻找一种新的方法。方法采用多向诱导、流式细胞术、细胞免疫荧光等方法对分离培养的细胞进行鉴定。分别在含Ong/ml和50ng/ml TNF-α的矿化诱导液中培养DPSCs,然后进行 Real-time PCR 和 Western blot 检测,观察 DPSCs 成牙/成骨分化及 BMP-4/Smad信号通路的变化。然后,对DPSCs的TSG-6基因进行修饰,使用Western blot和细胞免疫荧光技术,观察TSG-6基因修饰的DPSCs在成骨和炎症环境中BMP-4/Smad信号通路和成牙/成骨分化能力的变化。此外,将正常和TSG-6基因增强的DPSCs与注射用水凝胶混合移植于裸鼠皮下,6周后取材,以验证TSG-6是否影响牙髓干细胞的成牙/成骨分化。结果分离培养的细胞表达间充质干细胞标志蛋白Vimentin和Stro-1,但不表达肌上皮来源标志蛋白CK-14。流式细胞仪检测细胞表面分子,间充质干细胞标志物CD73、CD90均为阳性,血源性标记物CD31、上皮来源标志物CD34及T细胞标记物CD3均为阴性。实时荧光定量PCR和Western结果表明,高表达的TSG-6可以抑制Smad1/5磷酸化水平以及BMP-4、DMP-1、DSPP和ALP的表达;而炎症微环境中TSG-6敲低的DPSCs有更高的矿化能力。细胞免疫荧光显示矿化诱导后Ad-TSG-6 DPSCs矿化程度低于正常DPSCs;而在炎症环境中TSG-6-RNAi DPSCs的矿化程度高于正常DPSCs。此外,将正常和TSG-6修饰的DPSCs分别与注射用水凝胶混合植入裸鼠皮下,HE和MASSON染色结果显示正常的DPSCs形成了富含胶原纤维的组织,而Ad-TSG-6 DPSCs形成的皮下组织变异性大,细胞密度高。Ad-TSG-6 DPSCs的OCN、OPN、I型胶原、DMP-1、Ⅱ型胶原、BSP、RUNX2和DSPP等成牙/成骨标志物的表达均低于正常DPSCs。结论TNF-α通过升高TSG-6的表达抑制DPSCs中的BMP-4/smad信号通路及其成牙/成骨分化能力,降低TSG-6的表达可提高DPSCs在炎症微环境中的矿化能力,该结果可为提高DPSCs在炎症环境中的成牙本质向分化以及牙髓炎的治疗提供新的启示。更多还原

【Abstract】 BackgroundThe extracellular microenvironment can affect the differentiation ability of mesenchymal stem cells,especially the inflammatory microenvironment.It was found that inflamed pulp stem cells can express tumor necrosis factor-inducible protein 6(TSG-6),which can inhibit the mineralization of mesenchymal stem cells.However,whether the inhibition of odonto/osteogenesis ability of DPSCs induced by tumor necrosis factor(TNF-α)is related to TSG-6 or not,should be studied.The present study considers factors that inhibit the differentiation ability of dental pulp stem cells in an inflammatory environment as the breakthrough point,which means finding a novel method for the treatment of pulp inflammation.MethodsOsteogenic and dipogenic induction,flow cytometry,cellular immunofluorescence were performed to identify cells.DPSCs were cultured in mineralization inducer separately containing Ong/ml and 50ng/ml TNF-α for Real-time PCR and for Western blot to observe odonto/osteogenic differentiation of DPSCs and the variation of BMP-4/Smad signal pathway.Then,the TSG-6 gene of DPSCs was amplified or knocked down.The variation of BMP-4/Smad signal pathway and odonto/osteogenic differentiation ability of the TSG-6 gene-modified DPSCs in osteogenic and inflammatory environments in vitro were observed by Western blot and Cellular immunofluorescence.Moreover,normal and modified DPSCs combined with hydrogel were used for subcutaneous implantation in nude mice to verify whether TSG-6 plays a role in the effect of the inflammatory microenvironment on dental pulp stem cells or not.ResultsCells expressed Vimentin and Stro-1 proteins but did not express CK-14.Flow cytometry indicated that cells were positive for CD73,CD90 while negative for CD31,CD34 and CD3.Real-time PCR and Western blot were showed that high concentration of TNF-α and high expression of TSG-6 inhibits the expression of Smad1/5,BMP-4,DMP-1,DSPP and ALP.However,knockout of TSG-6 gene reduced the inhibition of TNF-α.Immunofluorescence indicated Ad-TSG-6 DPSCs were lower than those of normal DPSCs after mineralization induction,and TSG-6-RNAi DPSCs were higher compared with the normal DPSCs.Furthermore,subcutaneous transplantation of normal and modified DPSCs combined with hydrogel in nude mice demonstrated that normal DPSCs were formed in the bone-like tissues and Ad-TSG-6 DPSCs were highly variable and the cells were very dense.The expression of OCN,OPN,Collagen Ⅰ,DMP-1,Collagen Ⅱ,BSP,RUNX2 and DSPP of Ad-TSG-6 DPSCs were lower than normal DPSCs.ConclusionTNF-α regulates the BMP-4/smad signaling pathway and the odonto/osteogenic differentiation ability of DPSCs by regulating the expression of TSG-6,which can provide a novel enlightenment for the treatment of pulpitis.更多还原