[基因敲低促进人骨髓间充质干细胞的成骨分化]
[Knockdown of promotes osteogenic differentiation of human bone marrow mesenchymal stem cells].
作者信息
Shuai Ting, Guo Yanyan, Lin Chunping, Hou Xiaomei, Jin Chanyuan
机构信息
Second Clinical Division, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digi-tal Medical Devices & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China.
Department of Stomatology, Shengli Clinical Medical College of Fujian Medical University, Fujian Provincial Hospital, Fuzhou University Affiliated Provincial Hospital, Fuzhou 350001, China.
出版信息
Beijing Da Xue Xue Bao Yi Xue Ban. 2025 Feb 18;57(1):7-12. doi: 10.19723/j.issn.1671-167X.2025.01.002.
OBJECTIVE
To initially investigate the function of neuronal pentraxin 1 () 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 (), alkaline phosphatase (), osteocalcin (), and , were detected on the basis of quantitative real-time polymerase chain reaction (qPCR) technology. In order to establish a stable -knockdown hBMSCs cell line, shRNA lentivirus was constructed and used to infect hBMSCs. ALP staining, alizarin red (AR) staining, and qPCR were employed to assess the impact of -knockdown on the osteogenic differentiation ability of hBMSCs.
RESULTS
The results showed that during the osteogenic differentiation of hBMSCs , the mRNA expression levels of osteogenic genes , and significantly increased compared with 0 d, while expression decreased markedly ( < 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 was over 60%. After knocking down in hBMSCs, RNA was extracted from both the -knockdown group (sh 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 and osterix () were significantly higher in the sh group compared with the shNC group ( < 0.01). ALP staining revealed a significantly deeper coloration in the sh 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 group compared with the shNC group at the end of 14 d of osteogenic induction.
CONCLUSION
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 could potentially serve as a therapeutic target for the treatment of osteogenic abnormalities, including osteoporosis.
目的
初步探究神经元五聚体蛋白1（）基因对人骨髓间充质干细胞（hBMSCs）成骨分化的作用。
方法
诱导hBMSCs进行成骨分化，然后在不同时间点，即0、3、7、10和14天收集RNA。基于定量实时聚合酶链反应（qPCR）技术检测与成骨分化相关的关键基因，包括 runt相关转录因子2（）、碱性磷酸酶（）、骨钙素（）和 的mRNA表达水平。为建立稳定的 -敲低hBMSCs细胞系，构建 shRNA慢病毒并用于感染hBMSCs。采用碱性磷酸酶（ALP）染色、茜素红（AR）染色和qPCR评估 -敲低对hBMSCs成骨分化能力的影响。
结果
结果显示，在hBMSCs成骨分化过程中，与0天相比，成骨基因 、 和 的mRNA表达水平显著升高，而随着成骨诱导期延长， 表达明显降低（<0.01）。慢病毒感染72小时后，qPCR结果表明 的敲低效率超过60%。在hBMSCs中敲低 后，从在常规增殖培养基中培养的 -敲低组（sh 组）和对照组（shNC组）中提取RNA。qPCR结果显示，与shNC组相比，sh 组中成骨相关基因 和osterix（）的表达水平显著更高（<0.01）。ALP染色显示，在成骨诱导7天结束时，sh 组的染色明显比shNC组深。AR染色表明，在成骨诱导14天结束时，与shNC组相比，sh 组中矿化结节明显增加。
结论
在hBMSCs成骨分化中发挥调节作用，发现其敲低可增强hBMSCs的成骨分化。这一发现意味着 可能作为治疗包括骨质疏松症在内的成骨异常的治疗靶点。
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