DMOG对间充质干细胞生物学特性影响的研究
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摘要
[目的]探讨二甲基乙二酰基甘氨酸(dimethyloxaloylglycine,DMOG)对间充质干细胞(mesenchymal stem cells,MSCs)生物学特性的影响。[方法]以DMOG作用于P4代MSCs,分为DMOG 0、20、40和80μmol·L~(-1)组,以CCK-8法、流式细胞术、Transwell迁移法和Real-time q PCR依次检测不同浓度的DMOG对MSCs增殖能力、细胞周期、迁移能力以及成骨、成脂、成软骨和成神经分化能力的影响。[结果]DMOG各浓度组细胞生长曲线均接近S型,与DMOG 0μmol·L~(-1)组比较,20μmol·L~(-1)对MSCs增殖能力具有促进作用,40μmol·L~(-1)促进作用最明显,差异具有统计学意义(P<0.05)。与DMOG0μmol·L~(-1)组比较,DMOG处理可提高处于S期+G2/M期的MSCs细胞比例,差异具有统计学意义(P<0.05)。与DMOG 0μmol·L~(-1)组比较,DMOG20μmol·L~(-1)时促迁移作用显著(P<0.05)。DMOG处理后,MSCs仍保留了原有的成骨、成脂、成软骨、成神经分化能力,DMOG 20μmol·L~(-1)可显著促进MSCs成骨和成软骨分化能力,抑制MSCs成脂分化能力,差异具有统计学意义(P<0.05),对MSCs成神经分化能力无显著影响(P>0.05)。[结论]不同浓度DMOG处理后,MSCs仍保留了原有的多向分化能力,其中20μmol·L~(-1)DMOG可促进MSCs成骨和成软骨分化能力,抑制其成脂分化能力,对其成神经分化能力无显著影响,同时可显著提高MSCs增殖、迁移能力。
[Objective] To investigate the effect of dimethyloxaloylglycine(DMOG) on mesenchymal stem cells(MSCs) biological behavior in vitro. [Methods]The P4 generation of MSCs were treated with DMOG, and were divided into DMOG 0μmol·L~(-1) group, DMOG 20μmol·L~(-1) group, DMOG 40μmol·L~(-1) group and DMOG 80μmol·L~(-1) group. The CCK-8 assay, flow cytometry, transwell method, Real-time q PCR were adopted to detect the effects of DMOG on the proliferation, cell cycle, migration, and differentiation of MSCs. [Results]The cell growth curve of every group was close to S type after DMOG treatment(P<0.05), compared with DMOG 0μmol·L~(-1), the 20μmol·L~(-1) processing began to promoted cell proliferation ability of MSCs( P<0.05),and the promotion effect of DMOG 40μmol·L~(-1) was the most obvious(P <0.05); cells proportion of S phase +G2/M phase in MSCs was improved with DMOG treatment(P<0.05); compared with DMOG 0μmol·L~(-1), the promotion effect of migration ability was statistically significant of DMOG 20 μmol·L~(-1) processing(P<0.05); MSCs still kept the original osteogenic, adipogenic, chondrogenic, neural differentiation abilities after treated with DMOG, 20 μmol·L~(-1) treatment could obviously promote osteogenic and chondrogenic differentiation abilities( P<0.05), suppress adipogenic differentiation ability(P<0.05).[Conclusion]After processing with different concentration of DMOG, MSCs still kept the original multipotent differentiation abilities. 20 μmol·L~(-1) DMOG treatment could obviously promote osteogenic and chondrogenic differentiation abilities, suppress adipogenic differentiation ability, and could improve the proliferation and migration abilities of MSCs.
[Objective] To investigate the effect of dimethyloxaloylglycine(DMOG) on mesenchymal stem cells(MSCs) biological behavior in vitro. [Methods]The P4 generation of MSCs were treated with DMOG, and were divided into DMOG 0μmol·L~(-1) group, DMOG 20μmol·L~(-1) group, DMOG 40μmol·L~(-1) group and DMOG 80μmol·L~(-1) group. The CCK-8 assay, flow cytometry, transwell method, Real-time q PCR were adopted to detect the effects of DMOG on the proliferation, cell cycle, migration, and differentiation of MSCs. [Results]The cell growth curve of every group was close to S type after DMOG treatment(P<0.05), compared with DMOG 0μmol·L~(-1), the 20μmol·L~(-1) processing began to promoted cell proliferation ability of MSCs( P<0.05),and the promotion effect of DMOG 40μmol·L~(-1) was the most obvious(P <0.05); cells proportion of S phase +G2/M phase in MSCs was improved with DMOG treatment(P<0.05); compared with DMOG 0μmol·L~(-1), the promotion effect of migration ability was statistically significant of DMOG 20 μmol·L~(-1) processing(P<0.05); MSCs still kept the original osteogenic, adipogenic, chondrogenic, neural differentiation abilities after treated with DMOG, 20 μmol·L~(-1) treatment could obviously promote osteogenic and chondrogenic differentiation abilities( P<0.05), suppress adipogenic differentiation ability(P<0.05).[Conclusion]After processing with different concentration of DMOG, MSCs still kept the original multipotent differentiation abilities. 20 μmol·L~(-1) DMOG treatment could obviously promote osteogenic and chondrogenic differentiation abilities, suppress adipogenic differentiation ability, and could improve the proliferation and migration abilities of MSCs.
引文
[1] Xu K, Xiao J, Zheng K, et al. Mi R-21/STAT3 signal isinvolved in odontoblast differentiation of human dentalpulp stem cells mediated by TNF-α[J].Cell Reprogram,2018,20(2):107-116.
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[12] Liu L, Yu Q, Fu S, et al. The CXCR4 antagonistAMD3100 promotes mesenchymal stem cell mobilizationin rats preconditioned with the hypoxia-mimicking agentcobalt chloride[J]. Stem Cells Dev, 2018, 27(7):466-478.
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[16]胡韶君,余勤,刘丽珍,等.HIF-1信号通路在介导DMOG动员MSCs中的作用[J].中国比较医学杂志,2015,25(1):9-14.HU Shaojun, YU Qin, LIU Lizhen, et al.Mechanism ofHIF-1 signaling pathway in mediating MSCs mobilizationwith DMOG[J]. Chinese Journal of Comparative Medicine,2015, 25(1):9-14.
[17] Hudecova S, Lencesova L, Csaderova L, et al. Chemi-cally mimicked hypoxia modulates gene expression andprotein levels of the sodium calcium exchanger in HEK293 cell line via HIF-1α[J]. Gen Physiol Biophys, 2011,30(2):196-206.
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[19] Neth P, Ciccarella M, Egea V, et al. Wnt signaling regu-lates the invasion capacity of human mesenchymal stemcells[J]. Stem Cells, 2006, 24(8):1892-1903.
[20] Kumar S, Ponnazhagan S. Mobilization of bone marrowmesenchymal stem cells in vivo augments bone healingin a mouse model of segmental bone defect[J].Bone, 2012,50(4):1012-1018.
[21] Deng J, Zou Z, Zhou T, et al. The mobilization of rat’smesenchymal stem cells into peripheral blood by LiCLand its potency differentiation[J]. Chinese Sci Bull, 2008,53(17):2632-2638.
[22] Sherr C J. The pezcoller lecture:cancer cell cycles re-visited[J]. Cancer Res, 2000, 60(14):3689-3695.
[23] Marchbank T, Mahmood A, Harten S, et al. Dimethyloxa-lyglycine stimulates the early stages of gastrointestinalrepair processes through VEGF-dependent mechanisms[J].Lab Invest, 2011, 91(12):1684-1694.
[24] Cristancho A G, Lazar M A. Forming functional fat:agrowing understanding of adipocyte differentiation[J]. NatRev Mol Cell Biol, 2011, 12(11):722-734.
[25] Takada I, Kouzmenko A P, Kato S. Wnt and PPARgam-ma signaling in osteoblastogenesis and adipogenesis[J]. NatRev Rheumatol, 2009, 5(8):442-447.
[26] Prestwich T C, Macdougald O A. Wnt/beta-catenin sig-naling in adipogenesis and metabolism[J]. Curr Opin CellBiol, 2007, 19(6):612-617.
[27] Hoshiba T, Kawazoe N, Chen G. The balance of os-teogenic and adipogenic differentiation in human mes-enchymal stem cells by matrices that mimic stepwise tis-sue development[J]. Biomaterials, 2012,33(7):2025-2031.
[28] Gimble J M, Nuttall M E. The relationship between adi-pose tissue and bone metabolism[J]. Clin Biochem, 2012,45(12):874-879.
[29] Taipaleenmaki H, Abdallah B M, Aldahmash A, et al.Wntsignalling mediates the cross-talk between bone mar-row derived pre-adipocytic and pre-osteoblastic cell pop-ulations[J]. Exp Cell Res, 2011, 317(6):745-756.
[30] Vanella L, Kim D H, Asprinio D, et al. HO-1 expres-sion increases mesenchymal stem cell-derived osteoblastsbut decreases adipocyte lineage[J]. Bone,2010,46(1):236-243.
[31] Yu Q, Liu L, Duan Y, et al. Wnt/β-catenin signalingregulates neuronal differentiation of mesenchymal stemcells[J]. Biochem Biophys Res Commun,2013,439(2):297-302.
[2] Lo W J, Lin C L, Chang Y C, et al. Total body irradia-tion tremendously impair the proliferation, differentiationand chromosomal integrity of bone marrow-derived mes-enchymal stromal stem cells[J]. Ann Hematol, 2018,97(4):697-707.
[3] Link D.Stem cells on the move[J].Nat Med, 2010, 16(10):1073-1074.
[4] Hoggatt J, Speth J M, Pelus L M. Concise review:sow-ing the seeds of a fruitful harvest:hematopoietic stemcell mobilization[J]. Stem Cells, 2013,31(12):2599-2606.
[5] Gimble J M, Zvonic S, Floyd Z E, et al. Playing withbone and fat[J]. J Cell Biochem, 2006, 98(2):251-266.
[6] Martin P J, Haren N, Ghali O, et al. Adipogenic RNAsare transferred in osteoblasts via bone marrow adipocytes-derived extracellular vesicles(EVs)[J].BMC Cell Biol, 2015,16:10.
[7] Stute N, Holtz K, Bubenheim M, et al. Autologous serumfor isolation and expansion of human mesenchymal stemcells for clinical use[J]. Exp Hematol, 2004, 32(12):1212-1225.
[8] Moroni L, Fornasari P M. Human mesenchymal stemcells:a bank perspective on the isolation, characterizationand potential of alternative sources for the regenerationof musculoskeletal tissues[J]. J Cell Physiol, 2013, 228(4):680-687.
[9] Yagi H, Soto-Gutierrez A, Parekkadan B, et al. Mes-enchymal stem cells:mechanisms of immunomodulationand homing[J]. Cell Transplant, 2010, 19(6):667-679.
[10]刘伟,余勤,刘丽珍,等.脯氨酸羟化酶抑制剂对小鼠间充质干细胞的动员作用[J].浙江中医药大学学报, 2013,37(12):1371-1376.LIU Wei,YU Qin, LIU Lizhen, et al.Effect of prolylhy-droxylase inhibitor on mobilization of mesenchymal stemcells in mice[J]. Journal of Zhejiang Chinese Medical U-niversity, 2013,37(12):1371-1376.
[11] Lin D, Zhou L, Wang B, et al. Overexpression of HIF-1αin mesenchymal stem cells contributes to repairinghypoxic-ischemic brain damage in rats[J].C R Biol, 2017,340(1):18-24.
[12] Liu L, Yu Q, Fu S, et al. The CXCR4 antagonistAMD3100 promotes mesenchymal stem cell mobilizationin rats preconditioned with the hypoxia-mimicking agentcobalt chloride[J]. Stem Cells Dev, 2018, 27(7):466-478.
[13] He Q, Wan C, Li G. Concise review:multipotent mes-enchymal stromal cells in blood[J].Stem Cells,2007, 25(1):69-77.
[14] Pitchford S C, Furze R C, Jones C P, et al. Differentialmobilization of subsets of progenitor cells from the bonemarrow[J]. Cell Stem Cell, 2009, 4(1):62-72.
[15] Rochefort G Y, Delorme B, Lopez A, et al. Multipoten-tial mesenchymal stem cells are mobilized into peripheralblood by hypoxia[J]. Stem Cells, 2006, 24(10):2202-2208.
[16]胡韶君,余勤,刘丽珍,等.HIF-1信号通路在介导DMOG动员MSCs中的作用[J].中国比较医学杂志,2015,25(1):9-14.HU Shaojun, YU Qin, LIU Lizhen, et al.Mechanism ofHIF-1 signaling pathway in mediating MSCs mobilizationwith DMOG[J]. Chinese Journal of Comparative Medicine,2015, 25(1):9-14.
[17] Hudecova S, Lencesova L, Csaderova L, et al. Chemi-cally mimicked hypoxia modulates gene expression andprotein levels of the sodium calcium exchanger in HEK293 cell line via HIF-1α[J]. Gen Physiol Biophys, 2011,30(2):196-206.
[18] Kelly D J, Mecinovic J, Arnold N, et al. Upregulation ofhypoxia-inducible factor by dimethyloxalylglycine(DMOG)increases neovascularization within ischemic myocardiumin a porcine coronary occlusion model[J].J Am Coll Car-diol, 2010, 55(10Suppl):A216.E2047.
[19] Neth P, Ciccarella M, Egea V, et al. Wnt signaling regu-lates the invasion capacity of human mesenchymal stemcells[J]. Stem Cells, 2006, 24(8):1892-1903.
[20] Kumar S, Ponnazhagan S. Mobilization of bone marrowmesenchymal stem cells in vivo augments bone healingin a mouse model of segmental bone defect[J].Bone, 2012,50(4):1012-1018.
[21] Deng J, Zou Z, Zhou T, et al. The mobilization of rat’smesenchymal stem cells into peripheral blood by LiCLand its potency differentiation[J]. Chinese Sci Bull, 2008,53(17):2632-2638.
[22] Sherr C J. The pezcoller lecture:cancer cell cycles re-visited[J]. Cancer Res, 2000, 60(14):3689-3695.
[23] Marchbank T, Mahmood A, Harten S, et al. Dimethyloxa-lyglycine stimulates the early stages of gastrointestinalrepair processes through VEGF-dependent mechanisms[J].Lab Invest, 2011, 91(12):1684-1694.
[24] Cristancho A G, Lazar M A. Forming functional fat:agrowing understanding of adipocyte differentiation[J]. NatRev Mol Cell Biol, 2011, 12(11):722-734.
[25] Takada I, Kouzmenko A P, Kato S. Wnt and PPARgam-ma signaling in osteoblastogenesis and adipogenesis[J]. NatRev Rheumatol, 2009, 5(8):442-447.
[26] Prestwich T C, Macdougald O A. Wnt/beta-catenin sig-naling in adipogenesis and metabolism[J]. Curr Opin CellBiol, 2007, 19(6):612-617.
[27] Hoshiba T, Kawazoe N, Chen G. The balance of os-teogenic and adipogenic differentiation in human mes-enchymal stem cells by matrices that mimic stepwise tis-sue development[J]. Biomaterials, 2012,33(7):2025-2031.
[28] Gimble J M, Nuttall M E. The relationship between adi-pose tissue and bone metabolism[J]. Clin Biochem, 2012,45(12):874-879.
[29] Taipaleenmaki H, Abdallah B M, Aldahmash A, et al.Wntsignalling mediates the cross-talk between bone mar-row derived pre-adipocytic and pre-osteoblastic cell pop-ulations[J]. Exp Cell Res, 2011, 317(6):745-756.
[30] Vanella L, Kim D H, Asprinio D, et al. HO-1 expres-sion increases mesenchymal stem cell-derived osteoblastsbut decreases adipocyte lineage[J]. Bone,2010,46(1):236-243.
[31] Yu Q, Liu L, Duan Y, et al. Wnt/β-catenin signalingregulates neuronal differentiation of mesenchymal stemcells[J]. Biochem Biophys Res Commun,2013,439(2):297-302.