LncRNA-7460调控炎症来源牙周膜干细胞成骨分化的研究
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摘要
目的:探讨长链非编码RNA(lncRNA)在牙周膜干细胞成骨分化过程中的调控作用。方法:运用实时荧光定量聚合酶链反应实验(q PCR)观察表达量在成骨分化前后发生明显变化的lncRNA,筛选得到成骨相关的lncRNA-7460。运用慢病毒分别上调、下调炎症来源牙周膜间充质干细胞(PPDLSCs)内的lncRNA-7460,q PCR检测成骨相关基因mRNA的变化,用碱性磷酸酶(ALP)染色、ALP活性测定、茜素红染色等方法验证PPLDSCs成骨状态。数据进行统计学处理。结果:在PPDLSCs中上调lncRNA-7460引起成骨相关基因的表达上升,ALP活性增高,形成更多的钙结节;下调lncRNA-7460导致成骨相关基因表达下降,细胞ALP活性和成骨结节形成能力降低。结论:lncRNA-7460可以在体外培养中促进PPDLSCs的成骨分化能力。
Objective: To investigate the regulation of Long none noding RNAs( lncRNAs) in the osteogenic differentiation of human periodontal ligament stem cells( h PDLSCs). Methods: Real-time quantitative PCR( q PCR) was used to observe the different expression of lncRNA in h PDLSCs before and after osteogenic differentiation. And then,osteogenic-related lncRNA,lncRNA-7460 was screened out. After using lentivirus to overexpress and knockdown lncRNA-7460 in h PDLSCs respectively. q PCR was used to detect the mRNA expression of osteogenesis related genes. ALP activity assay and alizarin red staining were used to verify the effect of lncRNA-7460 on PPDLSCs. Data were statistically processed. Results: Overexpressing lncRNA-7460 in PPDLSCs promoted the expression of osteogenic differentiation related genes,increased ALP activity and mineralized nodule formation. Downexpression of lncRNA-7460 downregulated the expression of osteogenic differentiation related genes,decreased ALP activity and mineralized nodule formation. Conclusion: LncRNA-7460 can promote osteogenic differentiation of PPDLSCs in vitro.
Objective: To investigate the regulation of Long none noding RNAs( lncRNAs) in the osteogenic differentiation of human periodontal ligament stem cells( h PDLSCs). Methods: Real-time quantitative PCR( q PCR) was used to observe the different expression of lncRNA in h PDLSCs before and after osteogenic differentiation. And then,osteogenic-related lncRNA,lncRNA-7460 was screened out. After using lentivirus to overexpress and knockdown lncRNA-7460 in h PDLSCs respectively. q PCR was used to detect the mRNA expression of osteogenesis related genes. ALP activity assay and alizarin red staining were used to verify the effect of lncRNA-7460 on PPDLSCs. Data were statistically processed. Results: Overexpressing lncRNA-7460 in PPDLSCs promoted the expression of osteogenic differentiation related genes,increased ALP activity and mineralized nodule formation. Downexpression of lncRNA-7460 downregulated the expression of osteogenic differentiation related genes,decreased ALP activity and mineralized nodule formation. Conclusion: LncRNA-7460 can promote osteogenic differentiation of PPDLSCs in vitro.
引文
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[2]Liu N,Shi S,Deng M,et al.High levels ofβ-catenin signaling reduce osteogenic differentiation of stem cells in inflammatory microenvironments through inhibition of the noncanonical Wnt pathway[J].J Bone Miner Res,2011,26(9):2082-2095.
[3]Kong X,Liu Y,Ye R,et al.GSK3βis a checkpoint for TNF-α-mediated impaired osteogenic differentiation of mesenchymal stem cells in inflammatory microenvironments[J].Biochim Biophys Acta,2013,1830(11):5119-5129.
[4]Huang Y,Zheng Y,Jia L,et al.Long Noncoding RNA H19 Promotes Osteoblast Differentiation Via TGF-β1/Smad3/HDAC Signaling Pathway by Deriving miR-675[J].Stem Cells,2015,33(12):3481-3492.
[5]Wang L,Wu F,Song Y,et al.Long noncoding RNA related to periodontitis interacts with miR-182 to upregulate osteogenic differentiation in periodontal mesenchymal stem cells of periodontitis patients[J].Cell Death Dis,2016,7(8):e2327.
[6]Nanci A,Bosshardt DD.Structure of periodontal tissues in health and disease[J].Periodontol 2000,2006,40:11-28
[7]冯晓珂,董岩,李治治,等.骨髓间充质干细胞复合脱细胞软骨和富血小板血浆的体内成骨实验[J].实用口腔医学杂志,2017,33(2):148-151.
[8]Zhu W,Liang M.Periodontal ligament stem cells:Current status,concerns,and future prospects[J].Stem Cells Int,2015,2015:972313.
[9]Lee JT,Bartolomei MS.X-inactivation,imprinting,and long noncoding RNAs in health and disease[J].Cell,2013,152(6):1308-1323.
[10]Guttman M,Donaghey J,Carey BW,et al.lincRNAs act in the circuitry controlling pluripotency and differentiation[J].Nature,2011,477(7364):295-300.
[11]Ng SY,Stanton LW.Long non-coding RNAs in stem cell pluripotency[J].Wiley Interdiscip Rev RNA,2013,4(1):121-128.
[12]rom UA,Shiekhattar R.Long noncoding RNAs usher in a new era in the biology of enhancers[J].Cell,2013,154(6):1190-1193.
[13]Wapinski O,Chang HY.Long noncoding RNAs and human disease[J].Trends Cell Biol,2011,21(6):354-361.
[14]苏花,周珅玥,郭新程,等.LncRNA H19与OSF发生及癌变的关系[J].实用口腔医学杂志,2017,33(2):235-238.
[15]Jia Q,Jiang W,Ni L.Down-regulated non-coding RNA(lncRNA-ANCR)promotes osteogenic differentiation of periodontal ligament stem cells[J].Arch Oral Biol,2015,60(2):234-241.
[16]Zhu L,Xu PC.Downregulated LncRNA-ANCR promotes osteoblast differentiation by targeting EZH2 and regulating Runx2 expression[J].Biochem Biophys Res Commun,2013,432(4):612-617.
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