腺病毒介导的人端粒酶反转录酶牙周膜细胞系的建立
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摘要
目的通过腺病毒法建立稳定表达外源性人端粒酶反转录酶(hTERT)基因的牙周膜细胞系,以构建腺病毒介导的高效、稳定的hTERT牙周膜细胞系。方法聚合酶链反应(PCR)法扩增hTERT基因编码序列,构建同源重组腺病毒质粒p Ad-pshuttle-cmv-h TERT,收集腺病毒颗粒后感染人牙周膜细胞,实时荧光定量PCR检测h TERT及成骨相关基因碱性磷酸酶、核心结合因子2、骨涎蛋白、骨钙素、骨桥素、Ⅰ型胶原蛋白m RNA的表达水平,茜素红染色观察成骨分化能力,CCK-8检测细胞增殖能力。结果成功构建了含h TERT基因的腺病毒颗粒并感染牙周膜细胞,感染细胞与正常牙周膜细胞形态相似;实时荧光定量PCR结果显示h TERT及成骨相关基因在感染细胞中高表达;茜素红染色显示牙周膜细胞系成骨能力强;CCK-8结果示牙周膜细胞系增殖能力强。结论通过腺病毒法成功建立了过表达h TERT的人牙周膜细胞系,其具有较强的成骨分化能力,这为研究牙周膜再生机制提供了一个理想的细胞系。
Objective This study aims to establish an effective and stable periodontal ligament cell line stably expressing human telomerase reverse transcriptase(hTERT) gene by using the adenovirus method.Methods Polymerase chain reaction(PCR) was used to amplify the full length of hTERT gene to construct recombinant adenovirus plasmid pAd-pshuttle-cmvhTERT.Packaged adenovirus particles were used for infection of human periodontal ligament cells.The expression levels of h TERT and osteogenic genes,such as alkaline phosphatase,Runt-related transcription factor 2,bone sialoprotein,osteocalcin,osteopontin,and collagen Ⅰ mRNA,were detected by quantitative real-time PCR(qRT-PCR).The ability of osteogenic differentiation was observed by alizarin red staining,and the cell proliferation was determined by CCK-8.Results Adenovirus particles containing the hTERT gene were successfully constructed and infected with periodontal ligament cells.The infected cells were similar to normal periodontal ligament cells.The qRT-PCR results showed that hTERT and osteogenesis-associated genes were highly expressed in the periodontal ligament cell lines constructed by adenoviruses.Alizarin red staining showed that the periodontal ligament cell line had strong osteogenic differentiation capability.CCK-8 showed that the periodontal ligament cell line had strong proliferation capability.Conclusion The human periodontal ligament cell line with high efficiency and stable expression of hTERT was established by the adenovirus method,thereby providing an ideal cell line for studying the mechanism of periodontal regeneration.
Objective This study aims to establish an effective and stable periodontal ligament cell line stably expressing human telomerase reverse transcriptase(hTERT) gene by using the adenovirus method.Methods Polymerase chain reaction(PCR) was used to amplify the full length of hTERT gene to construct recombinant adenovirus plasmid pAd-pshuttle-cmvhTERT.Packaged adenovirus particles were used for infection of human periodontal ligament cells.The expression levels of h TERT and osteogenic genes,such as alkaline phosphatase,Runt-related transcription factor 2,bone sialoprotein,osteocalcin,osteopontin,and collagen Ⅰ mRNA,were detected by quantitative real-time PCR(qRT-PCR).The ability of osteogenic differentiation was observed by alizarin red staining,and the cell proliferation was determined by CCK-8.Results Adenovirus particles containing the hTERT gene were successfully constructed and infected with periodontal ligament cells.The infected cells were similar to normal periodontal ligament cells.The qRT-PCR results showed that hTERT and osteogenesis-associated genes were highly expressed in the periodontal ligament cell lines constructed by adenoviruses.Alizarin red staining showed that the periodontal ligament cell line had strong osteogenic differentiation capability.CCK-8 showed that the periodontal ligament cell line had strong proliferation capability.Conclusion The human periodontal ligament cell line with high efficiency and stable expression of hTERT was established by the adenovirus method,thereby providing an ideal cell line for studying the mechanism of periodontal regeneration.
引文
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[11]Bruderer M,Richards RG,Alini M,et al.Role and regulation of RUNX2 in osteogenesis[J].Eur Cell Mater,2014,28:269-286.
[12]Mi HW,Lee MC,Fu E,et al.Highly efficient multipotent differentiation of human periodontal ligament fibroblasts induced by combined BMP4 and hTERT gene transfer[J].Gene Therapy,2011,18(5):452-461.
[13]Sodek J,Ganss B,McKee MD.Osteopontin[J].Crit Rev Oral Biol Med,2000,11(3):279-303.
[2]甄蕾,刘宏伟.人牙周膜干细胞体外诱导分化为神经元样细胞的实验研究[J].华西口腔医学杂志,2009,27(1):71-74.Zhen L,Liu HW.Differentiation of human periodontal ligament stem cells into neuron-like cells in vitro[J].West Chin J Stomatol,2009,27(1):71-74.
[3]刘娟,赵红宇,轩东英,等.人牙周膜细胞群多向分化潜能的实验研究[J].华西口腔医学杂志,2010,28(2):185-189.Liu J,Zhao HY,Xuan DY,et al.Differentiation characteristics of human periodontal ligament cell population in vitro[J].West Chin J Stomatol,2010,28(2):185-189.
[4]Kamata N,Fujimoto R,Tomonari M,et al.Immortalization of human dental papilla,dental pulp,periodontal ligament cells and gingival fibroblasts by telomerase reverse transcriptase[J].J Oral Pathol Med,2004,33(7):417-423.
[5]Moffatt-Jauregui CE,Robinson B,de Moya AV,et al.Establishment and characterization of a telomerase immortalized human gingival epithelial cell line[J].J Periodont Res,2013,48(6):713-721.
[6]Fujii S,Maeda H,Wada N,et al.Establishing and characterizing human periodontal ligament fibroblasts immortalized by SV40T-antigen and hTERT gene transfer[J].Cell Tissue Res,2006,324(1):117-125.
[7]Salvi GE,Mischler DC,Schmidlin K,et al.Risk factors associated with the longevity of multi-rooted teeth.Longterm outcomes after active and supportive periodontal therapy[J].J Clin Periodontol,2014,41(7):701-707.
[8]Gronthos S,Chen SQ,Wang CY,et al.Telomerase accelerates osteogenesis of bone marrow stromal stem cells by upregulation of CBFA1,osterix,and osteocalcin[J].J Bone Miner Res,2003,18(4):716-722.
[9]Wescott DC,Pinkerton MN,Gaffey BJ,et al.Osteogenic gene expression by human periodontal ligament cells under cyclic tension[J].J Dent Res,2007,86(12):1212-1216.
[10]Fujisawa R,Tamura M.Acidic bone matrix proteins and their roles in calcification[J].Front Biosci(Landmark Ed),2012,17:1891-1903.
[11]Bruderer M,Richards RG,Alini M,et al.Role and regulation of RUNX2 in osteogenesis[J].Eur Cell Mater,2014,28:269-286.
[12]Mi HW,Lee MC,Fu E,et al.Highly efficient multipotent differentiation of human periodontal ligament fibroblasts induced by combined BMP4 and hTERT gene transfer[J].Gene Therapy,2011,18(5):452-461.
[13]Sodek J,Ganss B,McKee MD.Osteopontin[J].Crit Rev Oral Biol Med,2000,11(3):279-303.