NELL1慢病毒载体转染人牙周膜干细胞对其成骨分化影响的实验研究
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摘要
背景：NELL1，一种与人颅缝早闭症（craniosynostosis,CS）颅缝过早融合相关的新型分泌型蛋白，能促进间充质干细胞的成骨分化。
目的：研究NELL1对人牙周膜干细胞（humanperiodontalligamentstemcells,hPDLSC）体外成骨分化的作用并初步探讨其作用机制。
方法：构建携带NELL1的慢病毒载体（lenti-NELL1），用以转染hPDLSCs，经矿化诱导液诱导培养后，通过检测成骨标志物碱性磷酸酶(alkalinephosphate,ALP)、骨钙素（osteocalcin,OCN）及钙沉积来评估NELL1对hPDLSCs的骨诱导作用。同时real-timePCR检测成骨重要的转录因子（Msx2、Runx2）mRNA的表达变化。实验以只携带报告基因EGFP的空载体lenti-EGFP转染的hPDLSCs为对照。
结果：WesternBlot及real-timePCR证实构建的lenti-NELL1转染hADSCs能成功表达有生物学活性的NELL1蛋白。与对照相比，lenti-NELL1组ALP、OCN、钙沉积及Msx2mRNA都显著提高（p＜0.01），而两组间Runx2mRNA无显著差异(p＞0.01)。结论：构建编码NELL1基因的慢病毒lenti-NELL1能够成功感染hPDLSCs并使之长期稳定表达有生物学效应的NELL1。NELL1能显著促进hPDLSCs的成骨分化，在诱导成骨分化时，除了作为Runx2的下游介质外NELL1还与Msx2存在密切联系，慢病毒介导的NELL1体外基因治疗可能是一种理想的实现牙周再生的治疗策略。
NELL1is a newly identified secreted protein involved in craniosynostosis and has been found to promote osteogenic differentiation of mesenchymal stem cells. The objective of this study was to investigate the effect of NELL1on osteogenic differentiation of human periodontal ligament stem cells (hPDLSC) and the potential underlying mechanism.
Materials and Methods:hPDLSCs underwent lentivirus-mediated NELL1transfection (lenti-NELL1) and markers of osteogenesis were assessed (alkaline phosphate [ALP], osteocalcin [OCN], and calcium deposition) to evaluate the effect of NELL1on the differentiation of these cells. Real-time PCR was employed to measure the mRNA expression of Msx2and Runx2, and lenti-enhanced green fluorescent protein (EGFP) served as a control.
Results:Western blot and real-time PCR analyses confirmed that lenti-NELL1-transfected hPDLSCs could express NELL1. When compared with the lenti-EGFP group, ALP, OCN, calcium deposition, and Msx2mRNA expression were markedly increased (P<0.01), but there was no significant difference in Runx2mRNA expression between the two groups (P>0.01).
Conclusion:hPDLSCs can be transfected by lenti-NELL1and can stably express NELL1. NELL1can promote the osteogenic differentiation of hPDLSCs, which may be related to the downregulation of Msx2expression, lentivirus-mediated NELL1transfection can be used during in vitro gene therapy for periodontal regeneration.
目的：研究NELL1对人牙周膜干细胞（humanperiodontalligamentstemcells,hPDLSC）体外成骨分化的作用并初步探讨其作用机制。
方法：构建携带NELL1的慢病毒载体（lenti-NELL1），用以转染hPDLSCs，经矿化诱导液诱导培养后，通过检测成骨标志物碱性磷酸酶(alkalinephosphate,ALP)、骨钙素（osteocalcin,OCN）及钙沉积来评估NELL1对hPDLSCs的骨诱导作用。同时real-timePCR检测成骨重要的转录因子（Msx2、Runx2）mRNA的表达变化。实验以只携带报告基因EGFP的空载体lenti-EGFP转染的hPDLSCs为对照。
结果：WesternBlot及real-timePCR证实构建的lenti-NELL1转染hADSCs能成功表达有生物学活性的NELL1蛋白。与对照相比，lenti-NELL1组ALP、OCN、钙沉积及Msx2mRNA都显著提高（p＜0.01），而两组间Runx2mRNA无显著差异(p＞0.01)。结论：构建编码NELL1基因的慢病毒lenti-NELL1能够成功感染hPDLSCs并使之长期稳定表达有生物学效应的NELL1。NELL1能显著促进hPDLSCs的成骨分化，在诱导成骨分化时，除了作为Runx2的下游介质外NELL1还与Msx2存在密切联系，慢病毒介导的NELL1体外基因治疗可能是一种理想的实现牙周再生的治疗策略。
NELL1is a newly identified secreted protein involved in craniosynostosis and has been found to promote osteogenic differentiation of mesenchymal stem cells. The objective of this study was to investigate the effect of NELL1on osteogenic differentiation of human periodontal ligament stem cells (hPDLSC) and the potential underlying mechanism.
Materials and Methods:hPDLSCs underwent lentivirus-mediated NELL1transfection (lenti-NELL1) and markers of osteogenesis were assessed (alkaline phosphate [ALP], osteocalcin [OCN], and calcium deposition) to evaluate the effect of NELL1on the differentiation of these cells. Real-time PCR was employed to measure the mRNA expression of Msx2and Runx2, and lenti-enhanced green fluorescent protein (EGFP) served as a control.
Results:Western blot and real-time PCR analyses confirmed that lenti-NELL1-transfected hPDLSCs could express NELL1. When compared with the lenti-EGFP group, ALP, OCN, calcium deposition, and Msx2mRNA expression were markedly increased (P<0.01), but there was no significant difference in Runx2mRNA expression between the two groups (P>0.01).
Conclusion:hPDLSCs can be transfected by lenti-NELL1and can stably express NELL1. NELL1can promote the osteogenic differentiation of hPDLSCs, which may be related to the downregulation of Msx2expression, lentivirus-mediated NELL1transfection can be used during in vitro gene therapy for periodontal regeneration.
引文
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