低氧对人牙周膜干细胞增殖和分化的影响
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摘要
目的:观察低氧预处理对人牙周膜干细胞(human periodontal ligament stem cells,hPDLSCs)干性的影响。方法:收集hPDLSCs并进行鉴定后,分别在常氧(20%O2)和低氧(5%O2)环境下培养5d,噻唑蓝(methyl thiazol tetrazolium,MTT)法检测细胞的增殖能力。分别在低氧环境培养1d和5d,实时定量聚合酶链反应(Real-time quantitative polymerase chain reaction,Real-time PCR)检测hPDLSCs中干性相关基因Oct4、Sox2、Nanog的mRNA表达水平。细胞增殖核抗原Ki-67染色法和结晶紫染色观察细胞自我更新能力。分别用成骨和成脂诱导hPDLSCs21d,观察低氧对人牙周膜干细胞多项分化能力的影响。结果:MTT结果显示低氧预处理能显著增强hPDLSCs增殖能力(P<0.05)。Real-time PCR结果显示低氧在第1天和第5天时能够提高细胞的干性基因表达(P<0.05),其中在第5天的时候SOX2的提高最为显著(P<0.05)。结晶紫染色结果表明,低氧环境能够增加细胞克隆形成率(P<0.05)。低氧环境还能增强干细胞的成骨分化能力,成骨相关基因RUNX2的表达显著提高(P<0.05),但是对成脂相关基因的增强不明显,对成脂标志基因PPARγ2的影响不明显。结论:低氧预处理hPDLSCs能够提高细胞的增殖和干性相关基因的表达。另外,低氧环境也能提高干细胞的分化,其中对成骨相关基因的促进作用较为显著,但对成脂分化影响不大。
Objective:To examine the effect of hypoxia on human periodontal ligament stem cells(hPDLSCs).Methods:HPDLSCs were collected and validated.Cells were cultured in normoxia(20%O_2)or hypoxia(5%O_2)for 5days.MTT was applied to detect the cell proliferation ability in each day.OCT4,SOX2,and NANOG genes were analyzed by RT-PCR in cells culture in hypoxia at day 1and day 5.Ki67 staining and crystal violet staining assay were used to detect the self-renewal ability of hPDLSCs.Osteogenic and adipogenic differentiation abilities were investigated after 21-day-induction.Results:The results from MTT assay showed that the proliferation ability of hPDLSCs treated with hypoxia was higher(P<0.05).Hypoxia treatment enhanced the related gene expression at both day 1and day 5(P<0.05).SOX2 was enhanced strongly at day 5(P<0.01).Crystal violet staining assay indicated that colony-forming efficiency was increased in hypoxia group(P<0.05).In addition,hypoxia also enhanced the osteogenic differentiation capacity of hPDLSCs.Osteogenic gene RUNX2 was significantly increased(P<0.05).However,although hypoxia increased the expression of adipogenic gene PPARγ2and adipogenic differentiation,the results was not statistically significant.Conclusion:Hypoxic preconditioning of hPDLSCs can increase cell proliferation and expression of stemness-related genes.In addition,hypoxia environment can also improve the osteogenesis differentiation of hPDLSCs,but has little effect on adipogenic differentiation.
Objective:To examine the effect of hypoxia on human periodontal ligament stem cells(hPDLSCs).Methods:HPDLSCs were collected and validated.Cells were cultured in normoxia(20%O_2)or hypoxia(5%O_2)for 5days.MTT was applied to detect the cell proliferation ability in each day.OCT4,SOX2,and NANOG genes were analyzed by RT-PCR in cells culture in hypoxia at day 1and day 5.Ki67 staining and crystal violet staining assay were used to detect the self-renewal ability of hPDLSCs.Osteogenic and adipogenic differentiation abilities were investigated after 21-day-induction.Results:The results from MTT assay showed that the proliferation ability of hPDLSCs treated with hypoxia was higher(P<0.05).Hypoxia treatment enhanced the related gene expression at both day 1and day 5(P<0.05).SOX2 was enhanced strongly at day 5(P<0.01).Crystal violet staining assay indicated that colony-forming efficiency was increased in hypoxia group(P<0.05).In addition,hypoxia also enhanced the osteogenic differentiation capacity of hPDLSCs.Osteogenic gene RUNX2 was significantly increased(P<0.05).However,although hypoxia increased the expression of adipogenic gene PPARγ2and adipogenic differentiation,the results was not statistically significant.Conclusion:Hypoxic preconditioning of hPDLSCs can increase cell proliferation and expression of stemness-related genes.In addition,hypoxia environment can also improve the osteogenesis differentiation of hPDLSCs,but has little effect on adipogenic differentiation.
引文
[1]董家辰,束蓉,宋忠臣.低氧与牙周再生相关细胞[J].口腔医学研究,2014,30(9):906-907.
[2]沈兰花,张瑞,孟玲娜.雌激素受体对去势大鼠牙周膜干细胞成骨分化能力的影响[J].口腔医学研究,2018,34(4):448-451.
[3]林文珍,高丽,牛晨光,等.人牙髓干细胞成骨分化前后相关基因的表达分析[J].口腔医学研究,2018,34(4):375-379.
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[6] Itoigawa Y,Kishimoto KN,Okuno H,et al.Hypoxia induces adipogenic differentitation of myoblastic cell lines[J].Biochem Biophys Res Commun,2010,399(4):721-726.
[7] Lee JS,Park JC,Kim TW,et al.Human bone marrow stem cells cultured under hypoxic conditions present altered characteristics and enhanced in vivo tissue regeneration[J].Bone,2015,78:34-45.
[8] Ahmed NE,Murakami M,Kaneko S,et al.The effects of hypoxia on the stemness properties of human dental pulp stem cells(DPSCs)[J].Sci Rep,2016,6:35476.
[2]沈兰花,张瑞,孟玲娜.雌激素受体对去势大鼠牙周膜干细胞成骨分化能力的影响[J].口腔医学研究,2018,34(4):448-451.
[3]林文珍,高丽,牛晨光,等.人牙髓干细胞成骨分化前后相关基因的表达分析[J].口腔医学研究,2018,34(4):375-379.
[4] Whitmill A,Liu Y,Timani KA,et al.Tip110deletion impaired embryonic and stem cell development involving downregulation of stem cell factors Nanog,Oct4,and Sox2[J].Stem Cells,2017,35(7):1674-1686.
[5] Wang Y,Xu Z,Jiang J,et al.Endogenous miRNA sponge lincRNA-RoR regulates Oct4,Nanog,and Sox2in human embryonic stem cell self-renewal[J].Dev Cell,2013,25(1):69-80.
[6] Itoigawa Y,Kishimoto KN,Okuno H,et al.Hypoxia induces adipogenic differentitation of myoblastic cell lines[J].Biochem Biophys Res Commun,2010,399(4):721-726.
[7] Lee JS,Park JC,Kim TW,et al.Human bone marrow stem cells cultured under hypoxic conditions present altered characteristics and enhanced in vivo tissue regeneration[J].Bone,2015,78:34-45.
[8] Ahmed NE,Murakami M,Kaneko S,et al.The effects of hypoxia on the stemness properties of human dental pulp stem cells(DPSCs)[J].Sci Rep,2016,6:35476.