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-cmv-hTERT. Packaged adenovirus particles were used for infection of human periodontal ligament cells. The expression levels of hTERT 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.
Conclusions: 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.
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pAd-pshuttle-cmv-hTERT，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hTERT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mRNA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hTERT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hTERT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hTERT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。.
Keywords: adenovirus; human telomerase reverse transcriptase; osteogenesis; periodontal ligament cells; stable expression.