【作者】 韦小浪； 陈文霞；

【Author】 Xiaolang Wei;Wenxia Chen;Department of Dentistry and Pulp,Affiliated Stomatological Hospital of Guangxi Medical University,Guangxi Key Laboratory for Prevention and Treatment of Oral infectious diseases;

【机构】 广西医科大学附属口腔医院牙体牙髓科,广西口腔感染性疾病防治重点实验室；

【摘要】 目的:探讨P物质/神经激肽受体1(Substance P/Neurokinin receptor1,SP/NK1R)信号轴对牙髓干细胞(Human dental pulp stem cells,hDPSCs)增殖、迁移和成牙本质向分化的影响。方法:体外常规培养和鉴定hDPSCs,在10-8M SP和hDPSCs共培养体系加入NK1R拮抗剂阿瑞匹坦(Aprepitant,Apr),将细胞分为SP组、SP+Apr组和对照组。通过CCK8实验、Transwell迁移实验、茜素红染色、碱性磷酸酶(Alkaline phosphatase,ALP)染色、RT-PCR和WB实验评估SP是否通过NK1R调控hDPSCs的增殖、迁移和成牙本质向分化。结果:CCK8结果显示96h时SP组的细胞增殖率明显高于对照组(P<0.05),加入Apr预处理后增殖率明显下降(P<0.05)。Transwell迁移实验显示,与对照组相比,SP组可明显促进hDPSCs的迁移,SP+Apr组抑制hDPSCs的迁移(P<0.05)。茜素红染色和ALP染色结果提示,SP组可轻度促进矿化结节的形成和ALP活性。RT-PCR结果显示,与对照组相比,SP组矿化相关因子ALP、I型胶原(Collagen typeⅠ,COLⅠ)、牙本质涎磷蛋白(Dentin sialophosphoprotein,DSPP)和Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)mRNA的表达量均上调(P<0.05);SP+Apr组相关因子mRNA的表达量均明显低于SP组(P<0.05)。WB结果显示,SP组RUNX2蛋白相对表达量高于对照组(P<0.05),而SP+Apr组DSPP、COL I和RUNX2蛋白表达量低于SP组,但差异均无统计学意义(P>0.05)。结论:SP通过与NK1R结合促进hDPSCs的增殖、迁移和成牙本质向分化。更多还原

【Abstract】 Objective:To investigate the effects of substance P/neurokinin receptor 1(SP/NK1R)signaling axis on the proliferation,migration,and odontogenic differentiation of human dental pulp stem cells(hDPSCs).Method:Routine in vitro culture and identification of hDPSCs were performed,and NK1R antagonist Apreptitant(Apr)was added to the co-culture system of 10-8M SP and hDPSCs.The cells were divided into SP group,SP+Apr group,and control group.Evaluate whether SP regulates the proliferation,migration,and odontogenic differentiation of hDPSCs through NK1R through CCK8 assay,Transwell migration assay,alizarin red staining,alkaline phosphatase(ALP) staining,RT-PCR,and WB assay.Result:CCK8 results showed that the cell proliferation rate of the SP group was significantly higher than that of the control group at 96 hours(P<0.05),the proliferation rate significantly decreased after adding Apr pretreatment(P<0.05).The Transwell migration experiment showed that compared with the control group,the SP group significantly promoted the migration of hDPSCs,while the SP+Apr group inhibited the migration of hDPSCs(P<0.05).The results of alizarin red staining and ALP staining suggest that the SP group can mildly promote the formation of mineralized nodules and ALP activity.The RT-PCR results showed that compared with the control group,the expression levels of mineralization related factors ALP,Collagen type I(COL I),Dentin sialophosphate protein(DSPP),and Runt related transcription factor 2(RUNX2) mRNA in the SP group were all upregulated(P<0.05);The expression level of mRNA related to factors in the SP+Apr group was significantly lower than that in the SP group(P<0.05).The WB results showed that the relative expression level of RUNX2 protein in the SP group was higher than that in the control group(P<0.05),while the expression levels of DSPP,COL I,and RUNX2 protein in the SP+Apr group were lower than those in the SP group,but the differences were not statistically significant(P>0.05).Conclusion:SP promotes the proliferation,migration,and odontogenic differentiation of hDPSCs by binding to NK1R.更多还原