Objective: To initially investigate the function of neuronal pentraxin 1 (NPTX1) gene on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).
Methods: hBMSCs were induced to undergo osteogenic differentiation, and then RNA was collected at different time points, namely 0, 3, 7, 10 and 14 d. The mRNA expression levels of key genes related with osteogenic differentiation, including runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), and NPTX1, were detected on the basis of quantitative real-time polymerase chain reaction (qPCR) technology. In order to establish a stable NPTX1-knockdown hBMSCs cell line, NPTX1 shRNA lentivirus was constructed and used to infect hBMSCs. ALP staining, alizarin red (AR) staining, and qPCR were employed to assess the impact of NPTX1-knockdown on the osteogenic differentiation ability of hBMSCs.
Results: The results showed that during the osteogenic differentiation of hBMSCs in vitro, the mRNA expression levels of osteogenic genes RUNX2, ALP and OCN significantly increased compared with 0 d, while NPTX1 expression decreased markedly (P < 0.01) as the osteogenic induction period exten-ded. At 72 h post-infection with lentivirus, the result of qPCR indicated that the knockdown efficiency of NPTX1 was over 60%. After knocking down NPTX1 in hBMSCs, RNA was extracted from both the NPTX1-knockdown group (sh NPTX1 group) and the control group (shNC group) cultured in regular proliferation medium. The results of qPCR showed that the expression levels of osteogenic-related genes RUNX2 and osterix (OSX) were significantly higher in the sh NPTX1 group compared with the shNC group (P < 0.01). ALP staining revealed a significantly deeper coloration in the sh NPTX1 group than in the shNC group at the end of 7 d of osteogenic induction. AR staining demonstrated a marked increase in mineralized nodules in the sh NPTX1 group compared with the shNC group at the end of 14 d of osteogenic induction.
Conclusion: NPTX1 exerts a modulatory role in the osteogenic differentiation of hBMSCs, and its knockdown has been found to enhance the osteogenic differentiation of hBMSCs. This finding implies that NPTX1 could potentially serve as a therapeutic target for the treatment of osteogenic abnormalities, including osteoporosis.
目的: 初步探究神经元正五聚体1(neuronal pentraxin 1，NPTX1)基因对人骨髓间充质干细胞(human bone marrow mesenchymal stem cells，hBMSCs)成骨分化调控的作用机制。
方法: 将hBMSCs进行成骨诱导培养，在不同的时间点(0、3、7、10、14 d)收集RNA，通过实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction，qPCR)技术检测成骨分化调控相关的Runt相关转录因子2(runt-related transcription factor 2，RUNX2)、碱性磷酸酶(alkaline phosphatase，ALP)、骨钙素(osteocalcin，OCN)以及NPTX1的mRNA表达水平。通过构建NPTX1 shRNA慢病毒并感染hBMSCs，建立NPTX1稳定敲减的hBMSCs细胞系；采用ALP染色、茜素红(alizarin red，AR)染色和qPCR等方法检测敲减NPTX1对hBMSCs成骨分化能力的影响。
结果: 体外诱导hBMSCs成骨分化时，随着成骨诱导时间的延长，与第0天相比，成骨基因RUNX2、ALP和OCN表达量均显著升高，而NPTX1表达量明显降低(P < 0.01)。慢病毒感染hBMSCs 72 h后，qPCR结果显示NPTX1敲减效率高于60%。hBMSCs敲减NPTX1后，将敲减NPTX1组(sh NPTX1组)及其对照组(shNC组)在普通增殖培养基下进行培养后提取RNA，qPCR结果显示和shNC组相比，sh NPTX1组的成骨相关基因RUNX2和成骨细胞特异性转录因子(osterix，OSX)表达量显著增高(P < 0.01)。ALP染色试验显示成骨诱导7 d时，sh NPTX1组较shNC组显色明显加深；AR染色试验显示成骨诱导14 d时，sh NPTX1组较shNC组矿化结节明显增多。
结论: NPTX1对hBMSCs成骨分化具有调控作用，且其敲减可促进hBMSCs的成骨分化，该结果提示NPTX1可能成为治疗骨质疏松症等成骨异常疾病的潜在靶点。
Keywords: Bone marrow mesenchymal stem cells; NPTX1 gene; Osteogenic differentiation.