中国组织工程研究

中国组织工程研究 ›› 2020, Vol. 24 ›› Issue (31): 4921-4926.doi: 10.3969/j.issn.2095-4344.2117

• 骨髓干细胞 bone marrow stem cells •    下一篇

孤儿核受体Rev-erbα在小鼠骨髓间充质干细胞成骨分化中的作用

张  霜1，2，徐晓梅1，2，曾  阳1，2，袁小平1，2，林富伟1，2    

  1. 1西南医科大学附属口腔医院正畸科，四川省泸州市  646000；2西南医科大学口颌面修复重建与再生实验室，四川省泸州市  646000

  • 收稿日期:2019-10-21 修回日期:2019-10-24 接受日期:2019-12-26 出版日期:2020-11-08 发布日期:2020-09-03
  • 通讯作者: 林富伟，硕士，主治医师，西南医科大学附属口腔医院正畸科，四川省泸州市 646000；西南医科大学口颌面修复重建与再生实验室，四川省泸州市 646000 袁小平，教授，主任医师，西南医科大学附属口腔医院正畸科，四川省泸州市646000；西南医科大学口颌面修复重建与再生实验室，四川省泸州市646000
  • 作者简介:张霜，女，1995年生，四川省成都市人，汉族，西南医科大学口腔医学院在读硕士，主要从事口腔临床医学研究。
  • 基金资助:
    四川省医学会项目(S18002)；泸州市科技局基金资助计划(2015-S-50(2/3))；西南医科大学附属口腔医院青年课题(201816)

Rev-erbα’s effect on osteoblastogenesis of mouse bone marrow mesenchymal stem cells 

Zhang Shuang1, 2, Xu Xiaomei1, 2, Zeng Yang1, 2, Yuan Xiaoping1, 2, Lin Fuwei1, 2    

  1. 1Department of Orthodontics, the Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; 2Oral & Maxillofacial Reconstruction and Regeneration Laboratory, Southwest Medical University, Luzhou 646000, Sichuan Province, China

  • Received:2019-10-21 Revised:2019-10-24 Accepted:2019-12-26 Online:2020-11-08 Published:2020-09-03
  • Contact: Lin Fuwei, Master, Attending physician, Department of Orthodontics, the Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; Oral & Maxillofacial Reconstruction and Regeneration Laboratory, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • About author:Zhang Shuang, Master candidate, Department of Orthodontics, the Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; Oral & Maxillofacial Reconstruction and Regeneration Laboratory, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Supported by:
    the Project of Sichuan Medical Association, No. S18002; the Fund of Luzhou Municipal Department of Science and Technology, No. 2015-S-50(2/3) ; the Youth Foundation of the Affiliated Stomatological Hospital of Southwest Medical University, No. 201816

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摘要：

文题释义：

近日节律基因：是对维持生物体昼夜节律起关键性作用的节律基因，受生物钟控制，且表达量随昼夜循环而发生节律性变化，通过振荡器系统形成反馈调节环路而调控生物节律。近日节律基因控制着一系列生理活动，包括觉醒周期、内分泌、心血管活动、体温、胃肠道蠕动以及能量代谢等。

Rev-erbα：由甲状腺受体α(TRα)的反义链编码，是重要的近日节律基因，将昼夜节律与新陈代谢通路的转录调控联系起来。Rev-erbα是一种强大的转录抑制因子，在哺乳动物的核心分子节律中发挥重要作用，同时也是肝脏和棕色脂肪组织等代谢组织中钟基因输出的关键调节因子。

背景：孤儿核受体Rev-erbα已被证明在骨代谢过程中发挥着重要作用，但其在调节骨髓间充质干细胞成骨分化过程中的具体机制尚不明确。

目的：研究孤儿核受体Rev-erbα是否参与小鼠骨髓间充质干细胞成骨分化能力的调节。

方法：以全骨髓贴壁法进行小鼠骨髓间充质干细胞的分离培养，构建携带Rev-erbα基因的慢病毒载体并转染至体外培养的第3代骨髓间充质干细胞，转染后48 h采用Real Time PCR法检测Rev-erbα基因水平的表达。实验分为3组：实验组骨髓间充质干细胞转染过表达Rev-erbα基因及EGFP基因的慢病毒载体，阳性对照组骨髓间充质干细胞转染含EGFP基因的空病毒载体，设立不含病毒原液的阴性对照组，分别进行成骨诱导并在第0，7，14天采用Real Time PCR法检测成骨分化相关指标碱性磷酸酶、骨桥蛋白、骨钙素的表达变化。

结果与结论：①携带Rev-erbα基因的慢病毒成功转染到骨髓间充质干细胞中并稳定表达；②随着成骨诱导时间延长，成骨相关指标碱性磷酸酶、骨桥蛋白表达增加，但组间差异无显著性意义，骨钙素表达增加，但实验组明显低于阳性对照组和对照组(P < 0.05)；③结果表明，Rev-erbα转染后骨髓间充质干细胞成骨分化能力降低，成骨晚期标志物骨钙素的表达受抑制，说明Rev-erbα在骨髓间充质干细胞成骨分化的晚期阶段有重要作用。

ORCID: 0000-0001-5227-1476(林富伟)

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 骨髓间充质干细胞, 成骨分化,  慢病毒,  Rev-erbα,  核受体

Abstract:

BACKGROUND: The orphan nuclear receptor Rev-erbα has been demonstrated to play important roles during bone metabolism. However, the specific mechanism underlying osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is still unclear.

OBJECTIVE: To explore whether the orphan nuclear receptor Rev-erbα participates in the osteogenesis process of BMSCs in mice.

METHODS: Mouse BMSCs were isolated and cultured by the whole bone marrow adherence method. And the lentivirus vector carrying the Rev-erbα gene was constructed and then transfected into the BMSCs. Real-time PCR was conducted to detect the mRNA level of Rev-erbα at 48 hours after transfection. There were three groups in the experiment: an experimental group with BMSCs transfected with lentiviral vectors containing overexpressed Rev-erbα gene and EGFP gene, a positive control group with BMSCs transfected with empty lentiviral vectors containing only EGFP gene, and a negative control group. Then the BMSCs were induced to differentiate into osteoblasts after the transfection and we detected the mRNA level of alkaline phosphatase, osteopontin, and osteocalcin using real-time PCR at 0, 7, and 14 days after induction.

RESULTS AND CONCLUSION: The lentivirus vector carrying Rev-erbα gene was successfully transfected into BMSCs, and expressed stably. An increase in the mRNA levels of alkaline phosphatase and osteopontin was detected in each group when cultured in osteogenic medium; however, there was no significant difference between groups. The mRNA level of osteocalcin was also increased in each group, and moreover, the mRNA level in the experimental group was significantly lower than that in the two control groups (P < 0.05). These results indicate that Rev-erbα transfection reduces the osteogenic ability of BMSCs, and the expression of osteocalcin, a late-stage osteogenic marker, is inhibited, indicating that the late-stage osteogenesis of BMSCs may be influenced by Rev-erbα.

Key words: bone marrow mesenchymal stem cells,  osteogenesis,  lentivirus,  Rev-erbα,  nuclear receptor

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