小鼠骨髓间充质干细胞成骨分化中的核受体Rev-erbα及Rorα
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摘要
背景:孤儿核受体Rev-erbα及Rorα已经被广泛证实与糖代谢、脂肪代谢等密切相关,但与骨代谢的关系尚不明确。目的:探讨孤儿核受体Rev-erbα及Rorα在骨髓间充质干细胞成骨分化过程中的作用。方法:采用全骨髓贴壁法分离培养C57BL/6小鼠骨髓间充质干细胞,进行形态学观察和成脂、成骨诱导分化,采用油红O染色,茜素红染色对其分化能力进行鉴定。在骨髓间充质干细胞成骨诱导分化第0,7,14天,采用Real Time PCR及Western Blot分别检测孤儿核受体Rev-erbα及Rorα的m RNA及蛋白表达变化。结果与结论:(1)骨髓间充质干细胞形态以梭形为主,呈典型的漩涡样生长。油红O染色及茜素红染色呈阳性;(2)在成骨诱导的第0,7,14天,Rev-erbα的m RNA及蛋白表达均呈下降趋势,而Rorα的m RNA及蛋白表达均呈上升趋势,提示Rev-erbα及Rorα在骨髓间充质干细胞成骨诱导过程中可能发挥了一定的作用。
BACKGROUND: The orphan nuclear receptors Rev-erbα and Rorα play important roles in lipid metabolism and glucose metabolism. But it is unclear whether they are involved in bone metabolism. OBJECTIVE: To observe the expression of Rev-erbα and Rorα during the osteoblastogenesis process of bone marrow mesenchymal stem cells(BMSCs). METHODS: BMSCs were isolated and purified from C57 BL/6 mice by the whole bone marrow adherence method followed by morphology observations and then BMSCs were induced to differentiate into osteoblasts and adipocytes. We detected their differentiation abilities using oil red O staining and alizarin red staining, respectively. Real-time PCR and western blot assay were conducted to detect the m RNA and protein levels of Rev-erbα and Rorα in BMSCs cultured in the osteogenic medium for 0, 7, 14 days. RESULTS AND CONCLUSION: BMSCs from C57 BL/6 mice were mainly spindle-shaped and exhibited the swirl-like pattern of growth. Following induction, oil red O staining and alizarin red staining produced a positive reaction in these cells. Rev-erbα expression at both gene and protein levels decreased at 0, 7, 14 days after osteogenic induction, while Rorα expression increased at both gene and protein levels. These findings indicate that Rev-erbα and Rorα may participate in the osteoblastogenesis of BMSCs.
BACKGROUND: The orphan nuclear receptors Rev-erbα and Rorα play important roles in lipid metabolism and glucose metabolism. But it is unclear whether they are involved in bone metabolism. OBJECTIVE: To observe the expression of Rev-erbα and Rorα during the osteoblastogenesis process of bone marrow mesenchymal stem cells(BMSCs). METHODS: BMSCs were isolated and purified from C57 BL/6 mice by the whole bone marrow adherence method followed by morphology observations and then BMSCs were induced to differentiate into osteoblasts and adipocytes. We detected their differentiation abilities using oil red O staining and alizarin red staining, respectively. Real-time PCR and western blot assay were conducted to detect the m RNA and protein levels of Rev-erbα and Rorα in BMSCs cultured in the osteogenic medium for 0, 7, 14 days. RESULTS AND CONCLUSION: BMSCs from C57 BL/6 mice were mainly spindle-shaped and exhibited the swirl-like pattern of growth. Following induction, oil red O staining and alizarin red staining produced a positive reaction in these cells. Rev-erbα expression at both gene and protein levels decreased at 0, 7, 14 days after osteogenic induction, while Rorα expression increased at both gene and protein levels. These findings indicate that Rev-erbα and Rorα may participate in the osteoblastogenesis of BMSCs.
引文
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[16]Yang W,Kang X,Liu J,et al.Clock Gene Bmal1 Modulates Human Cartilage Gene Expression by Crosstalk With Sirt1.Endocrinology.2016;157(8):3096-3107.
[17]Lin F,Chen Y,Li X,et al.Over-expression of circadian clock gene Bmal1 affects proliferation and the canonical Wnt pathway in NIH-3T3 cells.Cell Biochem Funct.2013;31(2):166-172.
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[21]Samsonraj RM,Raghunath M,Nurcombe V,et al.Concise Review:Multifaceted Characterization of Human Mesenchymal Stem Cells for Use in Regenerative Medicine.Stem Cells Transl Med.2017;6(12):2173-2185.
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[23]Castro-Manrreza ME,Mayani H,Monroy-García A,et al.Human mesenchymal stromal cells from adult and neonatal sources:a comparative in vitro analysis of their immunosuppressive properties against T cells.Stem Cells Dev.2014;23(11):1217-1232.
[24]Ma S,Xie N,Li W,et al.Immunobiology of mesenchymal stem cells.Cell Death Differ.2014;21(2):216-225.
[25]Javazon EH,Colter DC,Schwarz EJ,et al.Rat marrow stromal cells are more sensitive to plating density and expand more rapidly from single-cell-derived colonies than human marrow stromal cells.Stem Cells.2001;19(3):219-225.
[26]Volk SW,Diefenderfer DL,Christopher SA,et al.Effects of osteogenic inducers on cultures of canine mesenchymal stem cells.Am J Vet Res.2005;66(10):1729-1737.
[27]Bellantuono I,Aldahmash A,Kassem M.Aging of marrow stromal(skeletal)stem cells and their contribution to age-related bone loss.Biochimica et Biophysica Acta.2009;1792(4):364-370.
[28]Colter DC,Class R,Di Girolamo CM,et al.Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow.Proc Natl Acad Sci U S A.2000;97(7):3213-3218.
[29]Mortezaee K,Pasbakhsh P,Ragerdi Kashani I,et al.Melatonin Pretreatment Enhances the Homing of Bone Marrow-derived Mesenchymal Stem Cells Following Transplantation in a Rat Model of Liver Fibrosis.Iran Biomed J.2016;20(4):207-216.
[30]张荣耀,毕晓娟,马艳,等.全骨髓法培养C57小鼠骨髓间充质干细胞的生物学特性[J].中国组织工程研究,2014,18(1):45-50.
[31]Khanabdali R,Saadat A,Fazilah M,et al.Promoting effect of small molecules in cardiomyogenic and neurogenic differentiation of rat bone marrow-derived mesenchymal stem cells.Drug Des Devel Ther.2015;10:81-91.
[32]Bugge A,Feng D,Everett LJ,et al.Rev-erbαand Rev-erbβcoordinately protect the circadian clock and normal metabolic function.Genes Dev.2012;26(7):657-667.
[33]Turek FW,Joshu C,Kohsaka A,et al.Obesity and metabolic syndrome in circadian Clock mutant mice.Science.2005;308(5724):1043-1045.
[34]Duez H,van der Veen JN,Duhem C,et al.Regulation of bile acid synthesis by the nuclear receptor Rev-erbalpha.Gastroenterology.2008;135(2):689-698.
[35]Galli C,Passeri G,Macaluso GM.Osteocytes and WNT:the mechanical control of bone formation.J Dent Res.2010;89(4):331-343.
[36]Thudi NK,Martin CK,Murahari S,et al.Dickkopf-1(DKK-1)stimulated prostate cancer growth and metastasis and inhibited bone formation in osteoblastic bone metastases.Prostate.2011;71(6):615-625.
[37]Kim JH,Liu X,Wang J,et al.Wnt signaling in bone formation and its therapeutic potential for bone diseases.Ther Adv Musculoskelet Dis.2013;5(1):13-31.
[38]Wang Y,Li YP,Paulson C,et al.Wnt and the Wnt signaling pathway in bone development and disease.Front Biosci(Landmark Ed).2014;19:379-407.
[39]Hosoi T.Cytokines in bone diseases.Wnt signal and excessive bone formation.Clin Calcium.2010;20:1526-1531.
[40]Caronia G,Wilcoxon J,Feldman P,et al.Bone morphogenetic protein signaling in the developing telencephalon controls formation of the hippocampal dentate gyrus and modifies fear-related behavior.J Neurosci.2010;30:6291-6301.
[41]Schulze J,Seitz S,Saito H,et al.Negative regulation of bone formation by the transmembrane Wnt antagonist Kremen-2.PLo S One.2010;5:e10309.
[42]Boland GM,Perkins G,Hall DJ,et al.Wnt 3a promotes proliferation and suppresses osteogenic differentiation of adult human mesenchymal stem cells.J Cell Biochem.2004;93(6):1210-1230.
[43]Gaur T,Lengner CJ,Hovhannisyan H,et al.Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression.J Biol Chem.2005;280(39):33132-33140.
[2]Tang Z,Wei J,Yu Y,et al.γ-Secretase inhibitor reverts the Notch signaling attenuation of osteogenic differentiation in aged bone marrow mesenchymal stem cells.Cell Biol Int.2016;40(4):439-447.
[3]Zhou S,Greenberger JS,Epperly MW,et al.Age-related intrinsic changes in human bone-marrow-derived mesenchymal stem cells and their differentiation to osteoblasts.Aging Cell.2008;7(3):335-343.
[4]Tan J,Xu X,Tong Z,et al.Decreased osteogenesis of adult mesenchymal stem cells by reactive oxygen species under cyclic stretch:a possible mechanism of age related osteoporosis.Bone Res.2015;3:15003.
[5]Liu W,Qi M,Konermann A,et al.The p53/mi R-17/Smurf1pathway mediates skeletal deformities in an age-related model via inhibiting the function of mesenchymal stem cells.Aging(Albany NY).2015;7(3):205-218.
[6]Wang C,Wang J,Li J,et al.KDM5A controls bone morphogenic protein 2-induced osteogenic differentiation of bone mesenchymal stem cells during osteoporosis.Cell Death Dis.2016;7(8):e2335.
[7]Owen TA,Aronow MS,Barone LM,et al.Pleiotropic effects of vitamin D on osteoblast gene expression are related to the proliferative and differentiated state of the bone cell phenotype:dependency upon basal levels of gene expression,duration of exposure,and bone matrix competency in normal rat osteoblast cultures.Endocrinology.1991;128(3):1496-1504.
[8]Oldberg A,Jirskog-Hed B,Axelsson S,et al.Regulation of bone sialoprotein m RNA by steroid hormones.J Cell Biol.1989;109(6 Pt 1):3183-3186.
[9]Smith EP,Boyd J,Frank GR,et al.Estrogen resistance caused by a mutation in the estrogen-receptor gene in a man.N Engl J Med.1994;331(16):1056-1061.
[10]Miyamoto S,Cooper L,Watanabe K,et al.Role of retinoic acid-related orphan receptor-alpha in differentiation of human mesenchymal stem cells along with osteoblastic lineage.Pathobiology.2010;77(1):28-37.
[11]Meyer T,Kneissel M,Mariani J,et al.In vitro and in vivo evidence for orphan nuclear receptor RORalpha function in bone metabolism.Proc Natl Acad Sci U S A.2000;97(16):9197-9202.
[12]Kojetin DJ,Burris TP.REV-ERB and ROR nuclear receptors as drug targets.Nat Rev Drug Discov.2014;13(3):197-216.
[13]Welch RD,Billon C,Valfort AC,et al.Pharmacological inhibition of REV-ERB stimulates differentiation,inhibits turnover and reduces fibrosis in dystrophic muscle.Sci Rep.2017;7(1):17142.
[14]Samsa WE,Vasanji A,Midura RJ,et al.Deficiency of circadian clock protein BMAL1 in mice results in a low bone mass phenotype.Bone.2016;84:194-203.
[15]Suyama K,Silagi ES,Choi H,et al.Circadian factors BMAL1and RORαcontrol HIF-1αtranscriptional activity in nucleus pulposus cells:implications in maintenance of intervertebral disc health.Oncotarget.2016;7(17):23056-23071.
[16]Yang W,Kang X,Liu J,et al.Clock Gene Bmal1 Modulates Human Cartilage Gene Expression by Crosstalk With Sirt1.Endocrinology.2016;157(8):3096-3107.
[17]Lin F,Chen Y,Li X,et al.Over-expression of circadian clock gene Bmal1 affects proliferation and the canonical Wnt pathway in NIH-3T3 cells.Cell Biochem Funct.2013;31(2):166-172.
[18]Friedenstein AJ,Deriglasova UF,Kulagina NN,et al.Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method.Exp Hematol.1974;2(2):83-92.
[19]Lennon DP,Haynesworth SE,Bruder SP,et al.Human and animal mesenchymal progenitor cells from bone marrow:identification of serum for optimal selection and proliferation.In Vitro Cell Dev Biol.1996;32:602-611.
[20]Zheng YH,Xiong W,Su K,et al.Multilineage differentiation of human bone marrow mesenchymal stem cells in vitro and in vivo.Exp Ther Med.2013;5(6):1576-1580.
[21]Samsonraj RM,Raghunath M,Nurcombe V,et al.Concise Review:Multifaceted Characterization of Human Mesenchymal Stem Cells for Use in Regenerative Medicine.Stem Cells Transl Med.2017;6(12):2173-2185.
[22]Castro-Manrreza ME,Montesinos JJ.Immunoregulation by mesenchymal stem cells:biological aspects and clinical applications.J Immunol Res.2015;2015:394917.
[23]Castro-Manrreza ME,Mayani H,Monroy-García A,et al.Human mesenchymal stromal cells from adult and neonatal sources:a comparative in vitro analysis of their immunosuppressive properties against T cells.Stem Cells Dev.2014;23(11):1217-1232.
[24]Ma S,Xie N,Li W,et al.Immunobiology of mesenchymal stem cells.Cell Death Differ.2014;21(2):216-225.
[25]Javazon EH,Colter DC,Schwarz EJ,et al.Rat marrow stromal cells are more sensitive to plating density and expand more rapidly from single-cell-derived colonies than human marrow stromal cells.Stem Cells.2001;19(3):219-225.
[26]Volk SW,Diefenderfer DL,Christopher SA,et al.Effects of osteogenic inducers on cultures of canine mesenchymal stem cells.Am J Vet Res.2005;66(10):1729-1737.
[27]Bellantuono I,Aldahmash A,Kassem M.Aging of marrow stromal(skeletal)stem cells and their contribution to age-related bone loss.Biochimica et Biophysica Acta.2009;1792(4):364-370.
[28]Colter DC,Class R,Di Girolamo CM,et al.Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow.Proc Natl Acad Sci U S A.2000;97(7):3213-3218.
[29]Mortezaee K,Pasbakhsh P,Ragerdi Kashani I,et al.Melatonin Pretreatment Enhances the Homing of Bone Marrow-derived Mesenchymal Stem Cells Following Transplantation in a Rat Model of Liver Fibrosis.Iran Biomed J.2016;20(4):207-216.
[30]张荣耀,毕晓娟,马艳,等.全骨髓法培养C57小鼠骨髓间充质干细胞的生物学特性[J].中国组织工程研究,2014,18(1):45-50.
[31]Khanabdali R,Saadat A,Fazilah M,et al.Promoting effect of small molecules in cardiomyogenic and neurogenic differentiation of rat bone marrow-derived mesenchymal stem cells.Drug Des Devel Ther.2015;10:81-91.
[32]Bugge A,Feng D,Everett LJ,et al.Rev-erbαand Rev-erbβcoordinately protect the circadian clock and normal metabolic function.Genes Dev.2012;26(7):657-667.
[33]Turek FW,Joshu C,Kohsaka A,et al.Obesity and metabolic syndrome in circadian Clock mutant mice.Science.2005;308(5724):1043-1045.
[34]Duez H,van der Veen JN,Duhem C,et al.Regulation of bile acid synthesis by the nuclear receptor Rev-erbalpha.Gastroenterology.2008;135(2):689-698.
[35]Galli C,Passeri G,Macaluso GM.Osteocytes and WNT:the mechanical control of bone formation.J Dent Res.2010;89(4):331-343.
[36]Thudi NK,Martin CK,Murahari S,et al.Dickkopf-1(DKK-1)stimulated prostate cancer growth and metastasis and inhibited bone formation in osteoblastic bone metastases.Prostate.2011;71(6):615-625.
[37]Kim JH,Liu X,Wang J,et al.Wnt signaling in bone formation and its therapeutic potential for bone diseases.Ther Adv Musculoskelet Dis.2013;5(1):13-31.
[38]Wang Y,Li YP,Paulson C,et al.Wnt and the Wnt signaling pathway in bone development and disease.Front Biosci(Landmark Ed).2014;19:379-407.
[39]Hosoi T.Cytokines in bone diseases.Wnt signal and excessive bone formation.Clin Calcium.2010;20:1526-1531.
[40]Caronia G,Wilcoxon J,Feldman P,et al.Bone morphogenetic protein signaling in the developing telencephalon controls formation of the hippocampal dentate gyrus and modifies fear-related behavior.J Neurosci.2010;30:6291-6301.
[41]Schulze J,Seitz S,Saito H,et al.Negative regulation of bone formation by the transmembrane Wnt antagonist Kremen-2.PLo S One.2010;5:e10309.
[42]Boland GM,Perkins G,Hall DJ,et al.Wnt 3a promotes proliferation and suppresses osteogenic differentiation of adult human mesenchymal stem cells.J Cell Biochem.2004;93(6):1210-1230.
[43]Gaur T,Lengner CJ,Hovhannisyan H,et al.Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression.J Biol Chem.2005;280(39):33132-33140.