LED红光上调MAPK信号促进炎性环境中人牙髓干细胞成骨/成牙本质分化  

LED red light up⁃regulates MAPK signal to promote osteogenic/odontogenic differentiation of human dental pulp stem cells in an inflammatory environment

作　　者：刘源 惠以宁 姜冰 郑艮子 王瑶 LIU Yuan;HUI Yining;JIANG Bing;ZHENG Genzi;WANG Yao(Department of Preventive Health Care,the Affiliated Stomatological Hospital of Southwest Medical University,Luzhou 646000,China;Institute of Stomatology,Southwest Medical University,Luzhou 6460000,China;Oral and Maxillofacial Reconstruction and Regeneration of Luzhou Key Laboratory,Luzhou 6460000,China;Southwest Medical University,Luzhou 6460000,China)

机构地区：[1]西南医科大学附属口腔医院预防保健科,四川泸州646000 [2]西南医科大学口腔医学研究所,四川泸州646000 [3]口颌面修复重建和再生泸州市重点实验室,四川泸州646000 [4]西南医科大学,四川泸州646000

出　　处：《口腔疾病防治》2023年第10期701-711,共11页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：四川省科技计划联合创新专项任务项目(2022YFS0634);四川省科研课题计划项目(S21015);西南医科大学校级课题项目(2021ZKMS013)。

摘　　要：目的探讨发光二极管(light-emitting diode,LED)红光对人牙髓干细胞(human dental pulp stem cells,hDPSCs)成骨/成牙本质分化的影响及机制。方法本实验研究已通过单位伦理委员会审查批准。组织块酶消化法培养hDPSCs,在0、1、5、10μg/mL脂多糖(lipopolysaccharide,LPS)刺激下,CCK-8法检测hDPSCs增殖,筛选LPS刺激浓度。设置CG组(矿化诱导)、LPS+CG组、LPS+CG+LED光照组(能量分别为2、4、6、8、10 J/cm^(2))。第7天进行碱性磷酸酶(alkaline phosphatase,ALP)染色及活性测定,实时荧光定量PCR检测ALP、成骨细胞特异性转录因子(osterix,OSX)、牙本质基质蛋白-1(dentin matrix protein-1,DMP-1)、牙本质涎磷蛋白(dentin sialo-phosphoprotein,DSPP)基因表达情况,第21天进行茜素红染色及钙结节定量分析,筛选最佳光照能量。设置LPS+CG组、LPS+CG+LED组(最佳能量),ELISA法检测第1、3、5、7天肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)及白介素-1β(interlenkin-1β,IL-1β)表达量。Western blot法检测细胞外调节蛋白激酶1/2(extracellular regulated protein kinases 1/2,ERK1/2)、c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)、p38、细胞外调节蛋白激酶5(extracellular regulated protein kinases 5,ERK5)及磷酸化蛋白表达。分别阻断ERK1/2、JNK、ERK5、p38通路后,Western blot法检测第7天ALP、OSX、DMP-1、DSPP蛋白表达。结果CCK-8结果显示10μg/mL LPS诱导下,在第5、7天hDPSCs增殖低于0、1、5μg/mL LPS组(P<0.05),后续选择10μg/mL作为LPS刺激浓度。ALP染色及活性,ALP、OSX、DMP-1、DSPP的基因表达水平及钙结节定量结果示LPS+CG+4 J/cm^(2)组均高于其他处理组(P<0.05)。4 J/cmLED红光上调MAPK信号促进炎性环境中人牙髓干细胞成骨/成牙本质分化LED红光促成骨/成牙本质分化能力最强,作为后续实验光照能量。ELISA结果显示第5、7天,LPS+CG+LED组的TNF-α与IL-1β的分泌表达量低于LPS+CG组(P<0.05)。Western blot结果显示4 J/cm^(2) LED红�Objective To study the effect of light-emitting diode(LED)red light on the osteogenic/odontogenic differentiation of human dental pulp stem cells(hDPSCs)and its mechanism were discussed.Methods This study has been reviewed and approved by the Ethics Committee.hDPSCs were cultured by tissue block enzyme digestion.The proliferative capacity of hDPSCs was detected by the CCK-8 at days 1,3,5 and 7 under stimulation with 0,1,5 and 10μg/mL lipopolysaccharide(LPS),and the LPS stimulatory concentration was screened.The CG group(mineralization induction),LPS+CG group,and LPS+CG+(2,4,6,8,and 10 J/cm^(2))LED red light groups were set.On day 7,alkaline phosphatase(ALP)staining and ALP activity were determined.Relative expression levels of the ALP,osterix(OSX),dentin matrix protein-1(DMP-1)and dentin sialophosphoprotein(DSPP)genes were measured by qRT-PCR.On day 21,alizarin red staining and calcium nodule quantitative determination were performed to screen the best light energy.The LPS+CG group and LPS+CG+LED group(optimal energy)were set up,and the secretion and expression levels of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were detected by ELISAs on days 1,3,5 and 7.The relative expression levels of the extracellular regulated protein kinases 1/2(ERK1/2),p38,c-Jun N-terminal kinase(JNK),and extracellular regulated protein kinases 5(ERK5)proteins and their phosphorylated proteins in the MAPK signaling path-way were detected by Western blots.After the pathway was blocked,the relative expression levels of the ALP,OSX,DMP-1,and DSPP proteins after LED red light irradiation on day 7 were detected by Western blots.Results CCK-8 assays showed that the proliferation of hDPSCs induced by 10μg/mL LPS was lower than that of the 0,1,and 5μg/mL groups on the 5th and 7th days(P<0.05),and 10μg/mL was selected as the LPS stimulatory concentration in the follow-up experiment.ALP staining,ALP activity,gene expression levels of ALP,OSX,DMP-1 and DSPP and calcium nodule quantification in the LPS+CG+4 J/cm^(2) group were hi

关 键 词：人牙髓干细胞 脂多糖 炎性环境 发光二极管 红光 成骨/成牙本质分化 MAPK信号通路 成骨细胞特异性转录因子 牙本质基质蛋白-1 牙本质涎磷蛋白 

分 类 号：R78[医药卫生—口腔医学]