脂多糖干预ob/ob小鼠骨髓间充质干细胞的成骨分化
|
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100μg/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 mR NA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。
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 mR NA 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.
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 mR NA 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.
引文
[1]闫美玲,夏丽莎,胡长平.代谢炎症与肥胖相关研究进展[J].中国动脉硬化杂志,2015,23(6):634-638.
[2]熊均平,孔亚阁,杨旭.肥胖与慢性牙周炎相关性临床研究[J].实用口腔医学杂志,2015,31(2):259-261.
[3]Scheller EL,Song J,Dishowitz MI,et al.Leptin functions peripherally to regulate differentiation of mesenchymal progenitor cells.Stem Cells.2010;28(6):1071-1080.
[4]Reseland JE,Syversen U,Bakke I,et al.Leptin is expressed in and secreted from primary cultures of human osteoblasts and promotes bone mineralization.J Bone Miner Res.2001;16(8):1426-1433.
[5]Bandow K,Maeda A,Kakimoto K,et al.Molecular mechanisms of the inhibitory effect of lipopolysaccharide(LPS)on osteoblast differentiation.Biochem Biophys Res Commun.2010;402(4):755-761.
[6]王海燕.脂多糖刺激单核巨噬细胞培养上清液对成骨细胞成骨特性的影响[J].中华口腔正畸学杂志,2011,18(2):92-96.
[7]Lawrence CB,Brough D,Knight EM.Obese mice exhibit an altered behavioural and inflammatory response to lipopolysaccharide.Dis Model Mech.2012;5(5):649-659.
[8]Zhang Y,Proenca R,Maffei M,et al.Positional cloning of the mouse obese gene and its human homologue.Nature.1994;372(6505):425-432.
[9]Sanigorski A,Cameron-Smith D,Lewandowski P,et al.Impact of obesity and leptin treatment on adipocyte gene expression in Psammomys obesus.J Endocrinol.2000;164(1):45-50.
[10]胥丹妮,林晓萍.脂肪因子对牙周炎症组织影响机制的研究[J].口腔医学,2017,37(10):932-936.
[11]苏媛,吕影涛,张雪洋,等.肥胖复合牙周炎大鼠骨骼肌中瘦素受体表达的组织学研究[J].口腔疾病防治,2017,25(12):774-778.
[12]Hamrick MW,Pennington C,Newton D,et al.Leptin deficiency produces contrasting phenotypes in bones of the limb and spine.Bone.2004;34(3):376-383.
[13]Ulevitch RJ,Tobias PS.Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin.Annu Rev Immunol.1995;13:437-457.
[14]Pober JS,Sessa WC.Evolving functions of endothelial cells in inflammation.Nat Rev Immunol.2007;7(10):803-815.
[15]Tominari T,Hirata M,Matsumoto C,et al.Polymethoxy flavonoids,nobiletin and tangeretin,prevent lipopolysaccharide-induced inflammatory bone loss in an experimental model for periodontitis.JPharmacol Sci.2012;119(4):390-394.
[16]唐琪,王仁飞,陈莉丽.实验性牙槽骨吸收动物模型的建立[J].浙江实用医学,2008,13(1):11-12,25.
[17]Zhou CC,Xiong QC,Zhu XX,et al.AFF1 and AFF4 differentially regulate the osteogenic differentiation of human MSCs.Bone Res.2017;5:17044.
[18]赵永杰,付至江,谭伟光,等.影响MSCs向成骨与成脂肪分化因素的研究进展[J].中国骨质疏松杂志,2015,21(10):1272-1276.
[19]Liu R,Liu J,Guo L,et al.Effect of different proportions of bone marrow mesenchymal stem cells and endothelial cells on osteogenesis.Zhonghua Kou Qiang Yi Xue Za Zhi.2015;50(11):675-680.
[20]Ducy P,Zhang R,Geoffroy V,et al.Osf2/Cbfa1:a transcriptional activator of osteoblast differentiation.Cell.1997;89(5):747-754.
[21]李庆庆,王大平,熊建义,等.Runx2重组慢病毒感染骨髓间充质干细胞并促进其成骨分化的研究[J].中国临床解剖学杂志,2015,33(3):311-315.
[22]Komori T.Regulation of bone development and extracellular matrix protein genes by RUNX2.Cell Tissue Res.2010;339(1):189-195.
[23]刘红,廖二元,伍贤平.骨钙素与代谢性骨病[J].国外医学(内分泌学分册),2004,24(4):239-240,249.
[24]张振宇,韩一生,毕龙,等.降钙素相关肽诱导SD大鼠骨髓间充质干细胞成骨分化的机制[J].现代生物医学进展,2010,10(24):4606-4611.
[2]熊均平,孔亚阁,杨旭.肥胖与慢性牙周炎相关性临床研究[J].实用口腔医学杂志,2015,31(2):259-261.
[3]Scheller EL,Song J,Dishowitz MI,et al.Leptin functions peripherally to regulate differentiation of mesenchymal progenitor cells.Stem Cells.2010;28(6):1071-1080.
[4]Reseland JE,Syversen U,Bakke I,et al.Leptin is expressed in and secreted from primary cultures of human osteoblasts and promotes bone mineralization.J Bone Miner Res.2001;16(8):1426-1433.
[5]Bandow K,Maeda A,Kakimoto K,et al.Molecular mechanisms of the inhibitory effect of lipopolysaccharide(LPS)on osteoblast differentiation.Biochem Biophys Res Commun.2010;402(4):755-761.
[6]王海燕.脂多糖刺激单核巨噬细胞培养上清液对成骨细胞成骨特性的影响[J].中华口腔正畸学杂志,2011,18(2):92-96.
[7]Lawrence CB,Brough D,Knight EM.Obese mice exhibit an altered behavioural and inflammatory response to lipopolysaccharide.Dis Model Mech.2012;5(5):649-659.
[8]Zhang Y,Proenca R,Maffei M,et al.Positional cloning of the mouse obese gene and its human homologue.Nature.1994;372(6505):425-432.
[9]Sanigorski A,Cameron-Smith D,Lewandowski P,et al.Impact of obesity and leptin treatment on adipocyte gene expression in Psammomys obesus.J Endocrinol.2000;164(1):45-50.
[10]胥丹妮,林晓萍.脂肪因子对牙周炎症组织影响机制的研究[J].口腔医学,2017,37(10):932-936.
[11]苏媛,吕影涛,张雪洋,等.肥胖复合牙周炎大鼠骨骼肌中瘦素受体表达的组织学研究[J].口腔疾病防治,2017,25(12):774-778.
[12]Hamrick MW,Pennington C,Newton D,et al.Leptin deficiency produces contrasting phenotypes in bones of the limb and spine.Bone.2004;34(3):376-383.
[13]Ulevitch RJ,Tobias PS.Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin.Annu Rev Immunol.1995;13:437-457.
[14]Pober JS,Sessa WC.Evolving functions of endothelial cells in inflammation.Nat Rev Immunol.2007;7(10):803-815.
[15]Tominari T,Hirata M,Matsumoto C,et al.Polymethoxy flavonoids,nobiletin and tangeretin,prevent lipopolysaccharide-induced inflammatory bone loss in an experimental model for periodontitis.JPharmacol Sci.2012;119(4):390-394.
[16]唐琪,王仁飞,陈莉丽.实验性牙槽骨吸收动物模型的建立[J].浙江实用医学,2008,13(1):11-12,25.
[17]Zhou CC,Xiong QC,Zhu XX,et al.AFF1 and AFF4 differentially regulate the osteogenic differentiation of human MSCs.Bone Res.2017;5:17044.
[18]赵永杰,付至江,谭伟光,等.影响MSCs向成骨与成脂肪分化因素的研究进展[J].中国骨质疏松杂志,2015,21(10):1272-1276.
[19]Liu R,Liu J,Guo L,et al.Effect of different proportions of bone marrow mesenchymal stem cells and endothelial cells on osteogenesis.Zhonghua Kou Qiang Yi Xue Za Zhi.2015;50(11):675-680.
[20]Ducy P,Zhang R,Geoffroy V,et al.Osf2/Cbfa1:a transcriptional activator of osteoblast differentiation.Cell.1997;89(5):747-754.
[21]李庆庆,王大平,熊建义,等.Runx2重组慢病毒感染骨髓间充质干细胞并促进其成骨分化的研究[J].中国临床解剖学杂志,2015,33(3):311-315.
[22]Komori T.Regulation of bone development and extracellular matrix protein genes by RUNX2.Cell Tissue Res.2010;339(1):189-195.
[23]刘红,廖二元,伍贤平.骨钙素与代谢性骨病[J].国外医学(内分泌学分册),2004,24(4):239-240,249.
[24]张振宇,韩一生,毕龙,等.降钙素相关肽诱导SD大鼠骨髓间充质干细胞成骨分化的机制[J].现代生物医学进展,2010,10(24):4606-4611.