p38丝裂原活化蛋白激酶对人乳牙牙髓干细胞成骨分化能力的影响
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摘要
目的:比较人乳牙牙髓干细胞(SHED)及牙髓干细胞(DPSC)增殖及成骨分化能力差异,探讨p38丝裂原活化蛋白激酶(MAPK)在人乳牙牙髓干细胞及牙髓干细胞中的表达及对干细胞成骨分化能力的影响。方法:有限稀释法分离培养SHED和DPSC,ELISA法检测IL-1β、TNF-α分泌水平;PCR检测PCNA及CCND-1表达;两种干细胞常规培养及成骨诱导7d后进行ALP染色、实时定量PCR检测Runx2、OCN基因表达。采用Western blot检测两种细胞成骨诱导7d后p38及磷酸化p38(p-p38)的表达。使用p38 MAPK特异性抑制剂SB203580干扰后Western blot验证抑制效果;在SB203580干扰后流式细胞仪检测两种细胞周期变化,Western blot检测细胞OCN、ALP表达。结果:SH ED的PCNA、CCND-1、Runx 2、OCN表达水平高于DPSC。SH ED的p38表达量高于DPSC;成骨诱导后两种PDLSCs中的p-p38表达均升高,但p38表达水平有所降低。SB203580干扰后SHED的增殖指数及OCN和ALP表达水平显著降低。结论:SHED的增殖能力及成骨分化能力高于DPSC,p38 MAPK在调控SH ED的生物学功能中发挥重要作用,抑制p38后SH ED的增殖及成骨分化能力也被显著抑制。
Objective: Comparison of proliferation and osteogenic differentiation of human dental pulp stem cells and dental pulp stem cells.To investigate the expression of p38 mitogen-activated protein kinase(MAPK) in human dental pulp stem cells and dental pulp stem cells and their effects on stem cell differentiation. Methods: Separation and culture of SHED and DPSC by limiting dilution method. We measured TNF-α and IL-1β secretion and m RNA expression level of SHED and DPSC. Detection of IL-1β, TNF-α, PCNA and CCND-1 expression by PCR. SHED and DPSC were cultured and induced by osteogenic induction for 7 days. We performed ALP staining and real-time quantitative PCR were used to detect Runx2 and OCN gene expression. With the use of WB, we compared the levels of p38、 p-p38 following culture in osteogenic medium for 7 d of SHED and DPSC. Western blot was used to verify the inhibitory effect after interference with p38 MAPK specific inhibitor SB203580. After SB203580 interference, SHED and DPSC cell cycle were detected by flow cytometry,and the expressions of OCN and ALP were detected by Western blot. Results: The secretion of TNF-α and IL-1β and the gene expression level of SHED were significantly higher than those of DPSC, and the expression levels of PCNA, CCND-1,Runx 2 and OCN were also higher than those of DPSC. The p38 expression of SHED is higher than DPSC. The expression of p-p38 was increased in both PDLSCs after osteogenic induction, but the expression level of p38 was decreased. The proliferation index of SHED and the expression levels of OCN and ALP were significantly decreased after SB203580 interference. Conclusion: The proliferative capacity and osteogenic differentiation ability of SHED is higher than that of DPSC.P38 MAPK plays an important role in regulating the biological function of SHED. The inhibition of SHED proliferation and osteogenic differentiation after p38 is also significantly inhibited.
Objective: Comparison of proliferation and osteogenic differentiation of human dental pulp stem cells and dental pulp stem cells.To investigate the expression of p38 mitogen-activated protein kinase(MAPK) in human dental pulp stem cells and dental pulp stem cells and their effects on stem cell differentiation. Methods: Separation and culture of SHED and DPSC by limiting dilution method. We measured TNF-α and IL-1β secretion and m RNA expression level of SHED and DPSC. Detection of IL-1β, TNF-α, PCNA and CCND-1 expression by PCR. SHED and DPSC were cultured and induced by osteogenic induction for 7 days. We performed ALP staining and real-time quantitative PCR were used to detect Runx2 and OCN gene expression. With the use of WB, we compared the levels of p38、 p-p38 following culture in osteogenic medium for 7 d of SHED and DPSC. Western blot was used to verify the inhibitory effect after interference with p38 MAPK specific inhibitor SB203580. After SB203580 interference, SHED and DPSC cell cycle were detected by flow cytometry,and the expressions of OCN and ALP were detected by Western blot. Results: The secretion of TNF-α and IL-1β and the gene expression level of SHED were significantly higher than those of DPSC, and the expression levels of PCNA, CCND-1,Runx 2 and OCN were also higher than those of DPSC. The p38 expression of SHED is higher than DPSC. The expression of p-p38 was increased in both PDLSCs after osteogenic induction, but the expression level of p38 was decreased. The proliferation index of SHED and the expression levels of OCN and ALP were significantly decreased after SB203580 interference. Conclusion: The proliferative capacity and osteogenic differentiation ability of SHED is higher than that of DPSC.P38 MAPK plays an important role in regulating the biological function of SHED. The inhibition of SHED proliferation and osteogenic differentiation after p38 is also significantly inhibited.
引文
[1] Miura M, Gronthos S, Zhao M, et al. SHED:stem cells from human exfoliated deciduous teeth[J]. Proc Natl Acad Sci U S A, 2003, 100(10):5807-5812
[2] Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells(DPSCs)in vitro and in vivo[J]. Proc Natl Acad Sci USA, 2000, 97(25):13625-13630
[3] Vishwanath VR, Nadig RR, Nadig R, et al. Differentiation ofisolated and characterized human dental pulp stem cells and stem cells from human exfoliated deciduous teeth:An in vitro study[J]. J Conserv Dent, 2013, 16(5):423-428
[4] Yu D, Zhao X, Cheng JZ, et al. Downregulated microRNA-488 enhances odontoblast differentiation of human dental pulp stem cells via activation of the p38 MAPK signaling pathway[J]. J Cell Physiol,2019, 234(2):1442-1451
[5] Wang BL, Wang Z, Nan X,et al. Downregulation of micro RNA-143-5p is required for the promotion of odontoblasts differentiation of human dental pulp stem cells through the activation of the mitogen-activated protein kinases 14-dependent p38 mitogen-activated protein kinases signaling pathway[J]. J CellPhysiol, 2019, 234(4):4840-4850
[6] Zhu Y, Shang L, Chen X, et al. Deciduous dental pulp stem cells are involved in osteoclastogenesis during physiologic root resorption[J]. J Cell Physiol, 2013, 228(1):207-215
[7] Yasui T,Mabuchi Y, Morikawa S, et al. Isolation of dental pulp stem cells with high osteogenic potential[J]. Inflamm Regen,2017,37:8
[8] Arana-Arguez VE, Delgado-Rizo V, Pizano-Martinez OE,et al. Inhibitors of MAPK pathway ERK1/2 or p38 prevent the IL-1(3-induced up-regulation of SRP72 autoantigen in Jurkat cells[J]. J Biol Chem, 2010, 285(43):32824-32833
[9] Taguchi T, Yanagi Y, Yoshimaru K, et al. Regenerative medicine using stem cells from human xfoliated deciduous teeth(SHED):a promising new treatment in pediatric surgery[J]. Surg Today, 2019, 49(4):316-322
[10] Dai YY, Ni SY, Ma K, et al. Stem cells from human exfoliated deciduous teeth correct the immune imbalance of allergic rhinitis via Treg cells in vivo and in vitro[J]. Stem Cell Res Ther, 2019, 10(1):39
[11] Lee JM, Kim HY, Park JS, et al. Developing palatal bone using human mesenchymal stem cell and stem cells from exfoliateddeciduous teeth cell sheets[J]. J Tissue Eng Regen Med,2019, 13(2):319-327
[12] Yamaza T, Kentaro A, Chen C, et al. Immunomodulatory properties of stem cells from human exfoliated deciduous teeth. Stem Cell Res Ther, 2010, 1(1):5
[13] Silva Fde S, Ramos RN, de Almeida DC, et al. Mesenchymal stem cells derived from human exfoliated deciduous teeth(SHEDs)induce immune modulatory profile in monocytederived dendritic cells[J]. PLoS One, 2014, 9(5):e98050
[14] You Z, Liu SP, Du J, et al. Advancements in MAPK signaling pathways and MAPK-targeted therapies for ameloblastoma:A review[J]. J Oral Pathol Med, 2019, 48(3):201-205
[15] Cai B, Seong KJ, Bae SW, et al. Water-Soluble ArginylDiosgenin Analog Attenuates Hippocampal Neurogenesis Impairment Through Blocking Microglial Activation Underlying NF-κB and JNK MAPK Signaling in Adult Mice Challenged by LPS[J]. Mol Neurobiol, 2019, doi:10. 1007/s12035-019-1496-1503
[16] Su Y, Zong S,Wei C,Song F, et al. Salidroside promotes rat spinal cord injury recovery by inhibiting inflammatory cytokine expression and NF-κB and MAPK signaling pathways[J]. J Cell Physiol, 2019, doi:10. 1002/jcp. 28124
[2] Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells(DPSCs)in vitro and in vivo[J]. Proc Natl Acad Sci USA, 2000, 97(25):13625-13630
[3] Vishwanath VR, Nadig RR, Nadig R, et al. Differentiation ofisolated and characterized human dental pulp stem cells and stem cells from human exfoliated deciduous teeth:An in vitro study[J]. J Conserv Dent, 2013, 16(5):423-428
[4] Yu D, Zhao X, Cheng JZ, et al. Downregulated microRNA-488 enhances odontoblast differentiation of human dental pulp stem cells via activation of the p38 MAPK signaling pathway[J]. J Cell Physiol,2019, 234(2):1442-1451
[5] Wang BL, Wang Z, Nan X,et al. Downregulation of micro RNA-143-5p is required for the promotion of odontoblasts differentiation of human dental pulp stem cells through the activation of the mitogen-activated protein kinases 14-dependent p38 mitogen-activated protein kinases signaling pathway[J]. J CellPhysiol, 2019, 234(4):4840-4850
[6] Zhu Y, Shang L, Chen X, et al. Deciduous dental pulp stem cells are involved in osteoclastogenesis during physiologic root resorption[J]. J Cell Physiol, 2013, 228(1):207-215
[7] Yasui T,Mabuchi Y, Morikawa S, et al. Isolation of dental pulp stem cells with high osteogenic potential[J]. Inflamm Regen,2017,37:8
[8] Arana-Arguez VE, Delgado-Rizo V, Pizano-Martinez OE,et al. Inhibitors of MAPK pathway ERK1/2 or p38 prevent the IL-1(3-induced up-regulation of SRP72 autoantigen in Jurkat cells[J]. J Biol Chem, 2010, 285(43):32824-32833
[9] Taguchi T, Yanagi Y, Yoshimaru K, et al. Regenerative medicine using stem cells from human xfoliated deciduous teeth(SHED):a promising new treatment in pediatric surgery[J]. Surg Today, 2019, 49(4):316-322
[10] Dai YY, Ni SY, Ma K, et al. Stem cells from human exfoliated deciduous teeth correct the immune imbalance of allergic rhinitis via Treg cells in vivo and in vitro[J]. Stem Cell Res Ther, 2019, 10(1):39
[11] Lee JM, Kim HY, Park JS, et al. Developing palatal bone using human mesenchymal stem cell and stem cells from exfoliateddeciduous teeth cell sheets[J]. J Tissue Eng Regen Med,2019, 13(2):319-327
[12] Yamaza T, Kentaro A, Chen C, et al. Immunomodulatory properties of stem cells from human exfoliated deciduous teeth. Stem Cell Res Ther, 2010, 1(1):5
[13] Silva Fde S, Ramos RN, de Almeida DC, et al. Mesenchymal stem cells derived from human exfoliated deciduous teeth(SHEDs)induce immune modulatory profile in monocytederived dendritic cells[J]. PLoS One, 2014, 9(5):e98050
[14] You Z, Liu SP, Du J, et al. Advancements in MAPK signaling pathways and MAPK-targeted therapies for ameloblastoma:A review[J]. J Oral Pathol Med, 2019, 48(3):201-205
[15] Cai B, Seong KJ, Bae SW, et al. Water-Soluble ArginylDiosgenin Analog Attenuates Hippocampal Neurogenesis Impairment Through Blocking Microglial Activation Underlying NF-κB and JNK MAPK Signaling in Adult Mice Challenged by LPS[J]. Mol Neurobiol, 2019, doi:10. 1007/s12035-019-1496-1503
[16] Su Y, Zong S,Wei C,Song F, et al. Salidroside promotes rat spinal cord injury recovery by inhibiting inflammatory cytokine expression and NF-κB and MAPK signaling pathways[J]. J Cell Physiol, 2019, doi:10. 1002/jcp. 28124