大鼠牙胚来源外胚间充质干细胞中p75NTR时钟节律性表达
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摘要
目的研究p75神经营养因子受体(p75NTR)、神经生长因子(NGF)及相关时钟节律基因clock、per1、per2、Bmal1在24 h内的表达情况。方法取SD大鼠孕18.5 d胎鼠牙胚组织,采用组织块法培养牙胚来源外胚间充质干细胞(EMSCs)。流式细胞仪进行EMSCs表面分子鉴定CD14、CD29、CD44、CD45、CD90、CD105、CD146、CD166和p75NTR,流式细胞仪分选出p75NTR阳性的EMSCs。取分选后的第3代进行实验,血清休克试验进行细胞时钟节律性的诱发,在诱发后的0、4、8、12、16、20、24 h进行细胞收集,提取RNA;RealTime-PCR检测p75NTR、NGF、clock、per1、per2、Bmal1在24 h内不同时间点的表达量。结果组织块法成功培养出牙胚来源的EMSCs,经流式细胞仪检测细胞阳性表达CD14(93.58%)、CD29(95.16%)、CD44(98.66%)、CD90(92.72%)、CD105(28.50%)、CD146(95.21%)、CD166(97.23%)和p75NTR(90.18%),阴性表达CD45(0.43%);流式细胞仪成功分选p75NTR阳性EMSCs,细胞形态呈更为均一的间充质干细胞形态;RealTime-PCR检测p75NTR、NGF、clock、per1、per2、Bmal1不同时间点的表达量,呈现出一定的时钟节律性。结论 p75NTR与NGF及相关时钟基因在牙胚来源的EMSCs中呈节律性表达,p75NTR有可能成为时间生物学在牙齿领域研究进展中的关键性生物节律调控基因。
Objective To study the expression of p75 neurotrophic factor receptor(p75NTR),nerve growth factor(NGF)and related clock rhythm gene clock,per1,per2 and Bmal1 in 24 h.Methods The tooth germ tissue was obtained in SD rats with 18.5 days of gestation,using tissue mass method cultured Ecm stem cells from tooth germ(EMSCs).The EMSCs surface molecules with CD14,CD29,CD44,CD45,CD90,CD105,CD146,CD166 and p75NTR were identified by flow cytometry.p75NTR positive ectomesenchymal stem cells were isolated by flow cytometry,and the third passage EMSCs after isolation were recruited in this study.The cells were collected at 0,4,8,12,16,20,24 h after induction,and RNA was extracted.RealTime-PCR detected the expression of p75NTR,NGF,clock,per1,per2,Bmal1 at different time points in 24 h.Results The ectomesenchymal stem cells were successfully cultured by tissue mass method.The Positive expression of CD14(93.58%),CD29(95.16%),CD44(98.66%),CD90(92.72%),CD105(28.50%),CD146(95.21%),CD166(97.23%)and p75NTR(90.18%),negative expression of CD45(0.43%)were detected by flow cytometry.The p75NTR positive ectomesenchymal stem cells were successfully isolated by flow cytometry,the p75NTR positive cell morphology was more homogeneous and mesenchymal stem cells like.The expression of p75NTR,NGF,clock,per1,per2,Bmal1 showed chronorhythmia at different time points in 24 h.and circadian genes are circadian expression in EMSCs originated from teeth germ,p75NTR may be a key role of circadian rhythm genes in tooth research area of chronobiological development.
Objective To study the expression of p75 neurotrophic factor receptor(p75NTR),nerve growth factor(NGF)and related clock rhythm gene clock,per1,per2 and Bmal1 in 24 h.Methods The tooth germ tissue was obtained in SD rats with 18.5 days of gestation,using tissue mass method cultured Ecm stem cells from tooth germ(EMSCs).The EMSCs surface molecules with CD14,CD29,CD44,CD45,CD90,CD105,CD146,CD166 and p75NTR were identified by flow cytometry.p75NTR positive ectomesenchymal stem cells were isolated by flow cytometry,and the third passage EMSCs after isolation were recruited in this study.The cells were collected at 0,4,8,12,16,20,24 h after induction,and RNA was extracted.RealTime-PCR detected the expression of p75NTR,NGF,clock,per1,per2,Bmal1 at different time points in 24 h.Results The ectomesenchymal stem cells were successfully cultured by tissue mass method.The Positive expression of CD14(93.58%),CD29(95.16%),CD44(98.66%),CD90(92.72%),CD105(28.50%),CD146(95.21%),CD166(97.23%)and p75NTR(90.18%),negative expression of CD45(0.43%)were detected by flow cytometry.The p75NTR positive ectomesenchymal stem cells were successfully isolated by flow cytometry,the p75NTR positive cell morphology was more homogeneous and mesenchymal stem cells like.The expression of p75NTR,NGF,clock,per1,per2,Bmal1 showed chronorhythmia at different time points in 24 h.and circadian genes are circadian expression in EMSCs originated from teeth germ,p75NTR may be a key role of circadian rhythm genes in tooth research area of chronobiological development.
引文
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[20] ZHENG B,ALBRECHT U,KAASIK K,et al. Nonredundant roles of the mPer1 and m Per2 genes in the mammalian circadian clock[J]. Cell,2001,105(5):683-694.
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[22] ZHUANG YF,LI J. Serum EGF and NGF levels of patients with brain injury and limb fracture[J]. Asian Pac J Trop Med,2013,6(5):383-386.
[23] LEE JY,JAHNG JW,KIM SM,et al. Simultaneous inferior alveolar nerve regeneration and osseointegration with a nerve growth factorsupplying implant:a preliminary study[J]. J Oral Maxillofac Surg,2015,73(3):410-423.
[24] LI C,RISNES S. SEM observations of Retzius lines and prism cross-striations in human dental enamel after different acid etching regimes[J].Arch Oral Biol,2004,49(1):45-52.
[25] LEE CF,PROFFIT WR. The daily rhythm of tooth eruption[J]. Am J Orthod Dentofacial Orthop,1995,107(1):38-47.
[26] FRY JR,CHEEK CC,PATERSON RL,et al. Long-wave cycles in the position of erupting human premolars[J]. Arch Oral Biol,2004,49(12):1007-1013.
[2] DENG MJ,JIN Y,SHI JN,et al. Multilineage differentiation of ectomesenchymal cells isolated from the first branchial arch[J]. Tissue Eng,2004,10(9/10):1597-1606.
[3] ZHANG J,DUAN X,ZHANG H,et al. Isolation of neural crest-derived stem cells from rat embryonic mandibular processes[J]. Biol Cell,2006,98(10):567-575.
[4] NIE X,XING Y,DENG M,et al. Ecto-mesenchymal stem cells from facial process:potential for muscle regeneration[J]. Cell Biochem Biophys,2014,70(1):615-622.
[5] WEN X,LIU L,DENG M,et al. Characterization of p75(+)ectomesenchymal stem cells from rat embryonic facial process tissue[J]. Biochem Biophys Res Commun,2012,427(1):5-10.
[6]赵曼竹,温秀杰,李刚,等. p75神经营养受体阳性外胚间充质干细胞的体外获取与生物学特性研究[J].中华口腔医学杂志,2015,50(2):103-109.
[7] WEN X,LIU L,DENG M,et al. In vitro cementoblast-like differentiation of postmigratory neural crest-derived p75(+)stem cells with dental follicle cell conditioned medium[J]. Exp Cell Res,2015,337(1):76-86.
[8]金岩,李玉成,史俊南.组织工程化牙齿研究:机遇与挑战[J].组织工程与重建外科,2006(1):1-4.
[9] YANG K,WANG Y,JU Y,et al. p75 neurotrophin receptor regulates differential mineralization of rat ectomesenchymal stem cells[J]. Cell Prolif,2016,50(1).
[10] SMITH TM. Experimental determination of the periodicity of incremental featuresinenamel[J].JAnat,2006,208(1):99-113.
[11] IINUMA Y,SUZUKI M,YOKOYAMA M,et al. Daily incremental lines in sika deer(Cervus nippon)dentine[J]. J Vet Med Sci,2002,64(9):791-795.
[12] YAMAMOTO T,DOMON T,TAKAHASHI S,et al. Twisted plywood structure of an alternating lamellar pattern in cellular cementum of human teeth[J]. Anat Embryol(Berl),2000,202(1):25-30.
[13] BAEZA-RAJA B,ECKEL-MAHAN K,ZHANG L,et al. p75 neurotrophin receptor is a clock gene that regulates oscillatory components of circadian and metabolic networks[J]. J Neurosci,2013,33(25):10221-10234.
[14] LOMEN-HOERTH C,SHOOTER EM. Widespread neurotrophin receptor expression in the immune system and other nonneuronal rat tissues[J].J Neurochem,1995,64(4):1780-1789.
[15] DIBNER C,SCHIBLER U,ALBRECHT U. The mammalian circadian timing system:organization and coordination of central and peripheral clocks[J]. Annu Rev Physiol,2010,72:517-549.
[16] ASHER G,SCHIBLER U. Crosstalk between components of circadian and metabolic cycles in mammals[J]. Cell Metab,2011,13(2):125-137.
[17] SOLT LA,WANG Y,BANERJEE S,et al. Regulation of circadian behaviour and metabolism by synthetic REV-ERB agonists[J]. Nature,2012,485(7396):62-68.
[18] SCHIBLER U,SASSONE-CORSI P. A web of circadian pacemakers[J].Cell,2002,111(7):919-922.
[19] BALSALOBRE A,DAMIOLA F,SCHIBLER U. A serum shock induces circadian gene expression in mammalian tissue culture cells[J]. Cell,1998,93(6):929-937.
[20] ZHENG B,ALBRECHT U,KAASIK K,et al. Nonredundant roles of the mPer1 and m Per2 genes in the mammalian circadian clock[J]. Cell,2001,105(5):683-694.
[21] YOO SH,KO CH,LOWREY PL,et al. A noncanonical E-box enhancer drives mouse Period2 circadian oscillations in vivo[J]. Proc Nati Acad Sci USA,2005,102(7):2608-2613.
[22] ZHUANG YF,LI J. Serum EGF and NGF levels of patients with brain injury and limb fracture[J]. Asian Pac J Trop Med,2013,6(5):383-386.
[23] LEE JY,JAHNG JW,KIM SM,et al. Simultaneous inferior alveolar nerve regeneration and osseointegration with a nerve growth factorsupplying implant:a preliminary study[J]. J Oral Maxillofac Surg,2015,73(3):410-423.
[24] LI C,RISNES S. SEM observations of Retzius lines and prism cross-striations in human dental enamel after different acid etching regimes[J].Arch Oral Biol,2004,49(1):45-52.
[25] LEE CF,PROFFIT WR. The daily rhythm of tooth eruption[J]. Am J Orthod Dentofacial Orthop,1995,107(1):38-47.
[26] FRY JR,CHEEK CC,PATERSON RL,et al. Long-wave cycles in the position of erupting human premolars[J]. Arch Oral Biol,2004,49(12):1007-1013.