中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (5): 703-709.doi: 10.3969/j.issn.2095-4344.1563

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

脂多糖干预ob/ob小鼠骨髓间充质干细胞的成骨分化

刘大勇1，周伟伟1，2，肖 蕊1，王梅蕊1，赵梦明1，贾 智1   

  1. 1天津医科大学口腔医院牙体牙髓科，天津市 300070；2首都医科大学附属北京口腔医院急诊综合诊疗中心，北京市 100069
  • 修回日期:2018-11-03 出版日期:2019-02-18 发布日期:2019-02-18
  • 通讯作者: 贾智，主任医师，天津医科大学口腔医院牙体牙髓科，天津市 300070
  • 作者简介:刘大勇，男，1972年生，河北省迁西县人，汉族，2011年首都医科大学毕业，博士，副主任医师，主要从事干细胞与牙周组织再生研究。
  • 基金资助:

    国家自然科学基金(81670953)，项目负责人：刘大勇

Lipopolysaccharides affect osteogenic differentiation of bone marrow mesenchymal stem cells derived from ob/ob mice

Liu Dayong1, Zhou Weiwei1, 2, Xiao Rui1, Wang Meirui1, Zhao Mengming1, Jia Zhi1   

  1. 1Department of Dental Endodontics, Tianjin Medical University School & Hospital of Stomatology, Tianjin 300070, China; 2Emergency Comprehensive Medical Center, Beijing Stomatological Hospital, Capital Medical University, Beijing 100069, China
  • Revised:2018-11-03 Online:2019-02-18 Published:2019-02-18
  • Contact: Jia Zhi, Chief physician, Department of Dental Endodontics, Tianjin Medical University School & Hospital of Stomatology, Tianjin 300070, China
  • About author:Liu Dayong, PhD, Associate chief physician, Department of Dental Endodontics, Tianjin Medical University School & Hospital of Stomatology, Tianjin 300070, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81670953 (to LDY)

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摘要：

文章快速阅读：

文题释义：
脂多糖：是革兰阴性菌的表面抗原物质，是许多噬菌体吸附的受体，其毒性成分主要为类脂质A。脂多糖只有当细菌死亡溶解或用人工方法破坏菌细胞后才释放出来，所以叫做内毒素，其位于细胞壁的最外层，覆盖于细胞壁的黏肽上。
Ob/ob小鼠：是瘦素基因(ob)纯合突变的小鼠，1950年由Ingalls成功繁育，其特征为肥胖、多食、高血糖和胰岛素抵抗，近年来发现瘦素与肥胖密切相关。

 

摘要
背景：肥胖是牙周炎及机体其他疾病的易感因素，因肥胖与瘦素密切相关，目前尚无通过瘦素基因突变小鼠骨髓间充质干细胞的分化情况探索相关机制的研究。  
目的：通过研究ob/ob遗传性肥胖小鼠骨髓间充质干细胞在脂多糖刺激下的成骨能力及成骨相关分子的表达，从分子及细胞水平探讨肥胖与成骨能力的相关机制。
方法：选用8周龄雄性肥胖ob/ob小鼠、8周龄雄性C57小鼠(由中国医学科学院实验动物研究所提供)各8只，取两组小鼠双侧股骨，采用全骨髓法分离培养纯化得到ob/ob小鼠骨髓间充质干细胞及C57小鼠骨髓间充质干细胞，加入含有100 μg/L脂多糖的成骨诱导液进行成骨诱导分化，通过碱性磷酸酶染色、茜素红染色和碱性磷酸酶活性检测观察比较两组小鼠骨髓间充质干细胞成骨能力。采用RT-PCR检测两组小鼠骨髓间充质干细胞中Alp、Runx2、Ocn、Nf-κb mRNA表达水平。
结果与结论：①成骨诱导1周后，与C57小鼠骨髓间充质干细胞比较，ob/ob小鼠骨髓间充质干细胞碱性磷酸酶染色减弱，碱性磷酸酶活性显著降低(P < 0.01)；成骨诱导液中添加脂多糖后，碱性磷酸酶活性较未添加脂多糖组显著下降(P < 0.01)，ob/ob小鼠骨髓间充质干细胞碱性磷酸酶活性亦较C57小鼠骨髓间充质干细胞显著降低(P < 0.05)；②成骨诱导2周后，与C57小鼠骨髓间充质干细胞比较，ob/ob小鼠骨髓间充质干细胞茜素红染成橘红色的矿化结节减少，而成骨诱导液中添加脂多糖后，两组骨髓间充质干细胞均未见矿化结节形成；③成骨诱导后，与C57小鼠骨髓间充质干细胞比较，ob/ob小鼠骨髓间充质干细胞中Alp、Runx2、Ocn mRNA表达降低(P < 0.05)；诱导液中加入脂多糖后，Alp、Runx2、Ocn表达较未添加脂多糖组均显著减少(P < 0.05)，且在ob/ob小鼠骨髓间充质干细胞中Ocn、Runx2表达较C57小鼠骨髓间充质干细胞明显降低(P < 0.05)；④结果表明，瘦素缺乏的肥胖小鼠骨髓间充质干细胞成骨向分化发生障碍。在低浓度脂多糖刺激下，ob/ob小鼠骨髓间充质干细胞的成骨分化能力较C57小鼠骨髓间充质干细胞下降更明显。


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
ORCID: 0000-0001-8511-2658(刘大勇)

关键词: 骨髓间充质干细胞, 脂多糖, 肥胖, ob/ob小鼠, C57小鼠, 成骨分化, 国家自然科学基金

Abstract:

BACKGROUND: Obesity is a predisposing factor for periodontitis and other diseases in the body. Increasing evidence has proved the close correlation between obesity and leptin. However, there is still no study on the differentiation of bone marrow mesenchymal stem cells from mice with a mutation in leptin gene. 
OBJECTIVE: To investigate the osteogenic differentiation and expression of osteogenic related molecules of bone marrow mesenchymal stem cells derived from ob/ob mice by stimulation of lipopolysaccharide, and to explore the correlation between obesity and osteogenic ability at molecular and cellular levels. 
METHODS: Eight 8-week-old male ob/ob mice with hereditary obesity were used as experimental group, and eight 8-week-old C57 mice were used as control group. All the mice were provided by the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College in China. Bone marrow mesenchymal stem cells were isolated from bilateral femurs of all the mice using whole bone marrow culture method, cultured and purified. The cells were then cultured in the osteoinduction medium containing 100 μg/L lipopolysaccharide. Alkaline phosphatase staining, alizarin red staining and alkaline phosphatase activity detection were utilized to compare the osteogenic ability of bone marrow mesenchymal stem cells between the two groups. RT-PCR was used to detect the mRNA levels of Alp, Runx2, Ocn and Nf-κb. 
RESULTS AND CONCLUSION: (1) After 1 week of osteogenic induction, alkaline phosphatase staining and activity in the experimental group significantly weakened compared with the control group (P < 0.01), and the addition of lipopolysaccharide further decreased the alkaline phosphatase activity (P < 0.01), especially in the experimental group (P < 0.01). (2) After 2 weeks of osteogenic induction, less mineralized nodes were observed in the experimental group than the control group. After the addition of lipopolysaccharide, no mineralized nodes formed in the two groups. (3) After osteogenic induction, the expression of Alp, Runx2, Ocn and Nf-κb was significantly decreased in the experimental group than the control group (P < 0.05), and the addition of lipopolysaccharide strongly decreased the gene expression (P < 0.05), especially in the experimental group (P < 0.05). To conclude, the osteogenic differentiation of bone marrow mesenchymal stem cells from obesity mice with leptin deficiency is impaired. Under the stimulation of lipopolysaccharide at low concentration, bone marrow mesenchymal stem cells from ob/ob mice have lower osteogenic ability than those from C57 mice. 


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

Key words: Bone Marrow, Mesenchymal Stem Cells, Lipopolysaccharides, Leptin, Mice, Obese, Cell Differentiation, Tissue Engineering

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