Hes1调控牙髓干细胞增殖及对成牙本质分化相关蛋白表达的影响
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摘要
目的探讨发状分裂相关增强子1(Hes1)对牙髓干细胞增殖及对成牙本质分化相关蛋白的表达影响。方法分离人牙髓干细胞,细胞转染小干扰RNA对照(siRNA control)和Hes1小干扰RNA(siRNA Hes1)分别记为阴性组和干扰组,同时以没有转染的细胞为对照组。RT-PCR和Western blot检测转染效果,噻唑蓝(MTT)检测细胞增殖,试剂盒检测碱性磷酸酶(ALP)活性,Western blot检测牙本质涎磷蛋白(DSPP)、骨钙素(OCN)蛋白表达。结果与对照组相比,阴性组Hes1 mRNA和蛋白水平无显著差异;干扰组Hes1 mRNA和蛋白水平明显降低;与对照组相比,阴性组细胞存活率无显著差异(P=0.999),干扰组细胞存活率明显降低(P=0.026)。与对照组相比,阴性组ALP活性无显著差异(P=0.800),干扰组ALP活性明显升高(P=0.004)。与对照组相比,阴性组DSPP、OCN水平无显著差异(t_(DSPP)=0.408,P_(DSPP)=0.704,t_(OCN)=0.271,P_(OCN)=0.800),干扰组DSPP、OCN水平明显升高(t_(DSPP)=5.543,P_(DSPP)=0.005,t_(OCN)=10.063,P_(OCN)=0.001)。结论干扰Hes1表达能够抑制牙髓干细胞增殖,提高碱性磷酸酶活性,促进分化相关蛋白表达。
Objective To investigate the effect of Hes1 gene on proliferation of dental pulp stem cells and the expression of related proteins in odontoblast differentiation. Methods Human dental pulp stem cells were isolated and transfected with siRNA control and Hes1 siRNA,respectively,as negative group and interfering group,the cells without transfection were used as control group. Transfection effects were detected by RT-PCR and Western blot,cell proliferation was detected by MTT,the alkaline phosphatase activity was detected by kit. Western blot were used to detect the expression of DSPP and OCN proteins. Results There was no significant difference in Hes1 mRNA and protein level between the negative group and the control group. Compared with the control group,the level of Hes1 mRNA and protein in the interfering group was significantly decreased. There was no significant difference in the survival rate of the negative group compared with the control group( P = 0. 999),and the survival rate in the interfering group was significantly lower( P = 0. 026). Compared with the control group,the ALP activity of the negative group was not significantly different( P = 0. 800),ALP activity in the interfering group was significantly increased( P = 0. 004). Compared with the control group,the levels of DSPP and OCN in the negative group were not significantly different( t_(DSPP)= 0. 408,P_(DSPP)= 0. 704,t_(OCN)= 0. 271,P_(OCN)= 0. 800),and the levels of DSPP and OCN in the interfering group were significantly increased( t_(DSPP)= 5. 543,P_(DSPP)=0. 005,t_(OCN)= 10. 063,P_(OCN)= 0. 001). Conclusion Interference of Hes1 expression could inhibit the proliferation of dental pulp stem cells,increase alkaline phosphatase activity,promote differentiation related protein expression.
Objective To investigate the effect of Hes1 gene on proliferation of dental pulp stem cells and the expression of related proteins in odontoblast differentiation. Methods Human dental pulp stem cells were isolated and transfected with siRNA control and Hes1 siRNA,respectively,as negative group and interfering group,the cells without transfection were used as control group. Transfection effects were detected by RT-PCR and Western blot,cell proliferation was detected by MTT,the alkaline phosphatase activity was detected by kit. Western blot were used to detect the expression of DSPP and OCN proteins. Results There was no significant difference in Hes1 mRNA and protein level between the negative group and the control group. Compared with the control group,the level of Hes1 mRNA and protein in the interfering group was significantly decreased. There was no significant difference in the survival rate of the negative group compared with the control group( P = 0. 999),and the survival rate in the interfering group was significantly lower( P = 0. 026). Compared with the control group,the ALP activity of the negative group was not significantly different( P = 0. 800),ALP activity in the interfering group was significantly increased( P = 0. 004). Compared with the control group,the levels of DSPP and OCN in the negative group were not significantly different( t_(DSPP)= 0. 408,P_(DSPP)= 0. 704,t_(OCN)= 0. 271,P_(OCN)= 0. 800),and the levels of DSPP and OCN in the interfering group were significantly increased( t_(DSPP)= 5. 543,P_(DSPP)=0. 005,t_(OCN)= 10. 063,P_(OCN)= 0. 001). Conclusion Interference of Hes1 expression could inhibit the proliferation of dental pulp stem cells,increase alkaline phosphatase activity,promote differentiation related protein expression.
引文
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[13]邹晓英,庄姮,岳林,等.DAPT对人牙髓细胞Notch信号通路和细胞增殖的影响[J].北京大学学报:医学版,2013,45(2):274-279.
[14]La Noce M,Paino F,Spina A,et al.Dental pulp stem cells:state of the art and suggestions for a true translation of research into therapy[J].J Dent,2014,42(7):761-768.
[15]Iohara K,Nakashima M,Ito M,et al.Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein 2[J].J Dental Res,2004,83(8):590-595.
[16]Prescott RS,Alsanea R,Fayad MI,et al.In vivo generation of dental pulp-like tissue by using dental pulp stem cells,a collagen scaffold,and dentin matrix protein 1 after subcutaneous transplantation in mice[J].J Endod,2008,34(4):421-426.
[17]关丽娜.低氧环境下Notch信号通路对人牙髓干细胞増殖和分化的影响[D].西安:第四军医大学,2015.
[18]Yu J,Deng Z,Shi J,et al.Differentiation of dental pulp stem cells into regular-shaped dentin-pulp complex induced by tooth germ cell conditioned medium[J].Tissue Eng,2006,12(11):3097-3105.
[19]Conde CM,Demarco FF,Casagrande L,et al.Influence of poly-Llactic acid scaffold's pore size on the proliferation and differentiation of dental pulp stem cells[J].Braz Dent J,2015,26(2):93-98.
[20]马逢乐,刘宝刚,王娟,等.ERK信号通路在人牙髓干细胞增殖与分化中的作用[J].牙体牙髓牙周病学杂志,2015,25(5):277-281.
[2]Paino F,Noce M,Tirino V,et al.Histone deacetylase inhibition with valproic acid downregulates osteocalcin gene expression in human dental pulp stem cells and osteoblasts:evidence for HDAC2 involvement[J].Stem Cells,2014,32(1):279-289.
[3]De Obaldia M E,Bell J J,Wang X,et al.T cell development requires constraint of the myeloid regulator C/EBP-[alpha]by the Notch target and transcriptional repressor Hes1[J].Nat Immunol,2013,14(12):1277-1284.
[4]Jeliazkova P,J9rs S,Lee M,et al.Canonical Notch2 signaling determines biliary cell fates of embryonic hepatoblasts and adult hepatocytes independent of Hes1[J].Hepatol,2013,57(6):2469-2479.
[5]Osathanon T,Nowwarote N,Manokawinchoke J,et al.b FGF and JAGGED1 regulate alkaline phosphatase expression and mineralization in dental tissue-derived mesenchymal stem cells[J].J Cell Biochem,2013,114(11):2551-2561.
[6]Almeida PN,Souza GT,de Souza CM,et al.Proposing the use of dental pulp stem cells as a suitable biological model of neurofibromatosis type1[J].Child's Nerv Syst,2015,31(1):7-13.
[7]Fusco S,Leone L,Barbati SA,et al.A CREB-Sirt1-Hes1 circuitry mediates neural stem cell response to glucose availability[J].Cell Rep,2016,14(5):1195-1205.
[8]黄飞,王小琴.DAPT对Notch2/hes1信号通路在庆大霉素所致大鼠肾损伤及修复过程中表达的影响[J].临床和实验医学杂志,2015,14(17):1401-1405.
[9]Tatullo M,Marrelli M,Shakesheff KM,et al.Dental pulp stem cells:function,isolation and applications in regenerative medicine[J].J Tissue Eng Regen Med,2015,9(11):1205-1216.
[10]马亮.Notch信号在牙髓损伤修复中的作用研究[D].西安:第四军医大学,2010.
[11]常思佳,邹晓英,庄姮,等.激活Notch信号通路可延迟人牙髓细胞的老化表现[J].北京大学学报:医学版,2014,46(1):5-11.
[12]张忠提,王洋,贾兴亚.牙乳头细胞对大鼠牙髓干细胞中Notch信号的影响[J].解剖科学进展,2012,18(6):505-508.
[13]邹晓英,庄姮,岳林,等.DAPT对人牙髓细胞Notch信号通路和细胞增殖的影响[J].北京大学学报:医学版,2013,45(2):274-279.
[14]La Noce M,Paino F,Spina A,et al.Dental pulp stem cells:state of the art and suggestions for a true translation of research into therapy[J].J Dent,2014,42(7):761-768.
[15]Iohara K,Nakashima M,Ito M,et al.Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein 2[J].J Dental Res,2004,83(8):590-595.
[16]Prescott RS,Alsanea R,Fayad MI,et al.In vivo generation of dental pulp-like tissue by using dental pulp stem cells,a collagen scaffold,and dentin matrix protein 1 after subcutaneous transplantation in mice[J].J Endod,2008,34(4):421-426.
[17]关丽娜.低氧环境下Notch信号通路对人牙髓干细胞増殖和分化的影响[D].西安:第四军医大学,2015.
[18]Yu J,Deng Z,Shi J,et al.Differentiation of dental pulp stem cells into regular-shaped dentin-pulp complex induced by tooth germ cell conditioned medium[J].Tissue Eng,2006,12(11):3097-3105.
[19]Conde CM,Demarco FF,Casagrande L,et al.Influence of poly-Llactic acid scaffold's pore size on the proliferation and differentiation of dental pulp stem cells[J].Braz Dent J,2015,26(2):93-98.
[20]马逢乐,刘宝刚,王娟,等.ERK信号通路在人牙髓干细胞增殖与分化中的作用[J].牙体牙髓牙周病学杂志,2015,25(5):277-281.