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

doi:10.3969/j.issn.2095-4344.1563

Abstract

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

CLC Number:

Liu Dayong, Zhou Weiwei, Xiao Rui, Wang Meirui, Zhao Mengming, Jia Zhi. Lipopolysaccharides affect osteogenic differentiation of bone marrow mesenchymal stem cells derived from ob/ob mice[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(5): 703-709.

Figures/Tables 2

2.1 骨髓间充质干细胞的培养与鉴定结果 2.1.1 成脂诱导及油红O染色结果在成脂诱导液的作用下，细胞增殖变慢，体积渐变大，由梭形渐变椭圆形。约4周后可看到部分细胞胞浆内有串珠样折光明显的脂滴，油红O染色呈红色，与C57小鼠骨髓间充质干细胞相比，ob/ob小鼠骨髓间充质干细胞成脂诱导4周后倒置显微镜下观察可见较多红染的脂滴，见图1。 2.1.2 碱性磷酸酶染色结果小鼠骨髓间充质干细胞在成骨诱导培养液作用下第1周行碱性磷酸酶染色，大体观可见培养皿底呈较均匀蓝色着色，见图2。倒置显微镜下随机选取视野，与未加入脂多糖比较，加入100 μg/L脂多糖刺激后蓝染着色减弱，且可发现ob/ob小鼠骨髓间充质干细胞碱性磷酸酶染色较C57小鼠骨髓间充质干细胞着色减弱，见图3。 2.1.3 茜素红染色结果小鼠骨髓间充质干细胞在矿化诱导培养液的作用下，开始生长缓慢，随后增殖加快，5-7 d完全融合，换液过程中可发现少量细胞漂浮死亡，加入脂多糖诱导的细胞漂浮现象明显。诱导2周左右大体观察可见培养皿底部有散在的沙粒样乳白色结节，大小不一，换液不能去除。茜素红染色后倒置显微镜下观察，C57小鼠骨髓间充质干细胞可见橘红色矿化结节，较多且均匀分布，而ob/ob小鼠骨髓间充质干细胞成骨矿化较差，只见散在橘红色结节，加入脂多糖的两组小鼠骨髓间充质干细胞成骨诱导后未见明显矿化结节，见图4。 2.1.4 碱性磷酸酶活性检测结果成骨诱导后的碱性磷酸酶活性比未诱导组显著升高，差异有显著性意义(P < 0.01，n=6)。与C57小鼠骨髓间充质干细胞相比，ob/ob小鼠骨髓间充质干细胞成骨诱导1周后碱性磷酸酶活性显著降低，差异有显著性意义(P < 0.05)；成骨诱导液中添加脂多糖诱导1周后，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)，见图5。 2.2 RT-PCR检测成骨相关因子的表达成骨诱导1周后，ob/ob小鼠骨髓间充质干细胞中Alp、Ocn、Runx2基因表达水平均低于C57小鼠骨髓间充质干细胞，差异有显著性意义(P < 0.05)，Nf-κb基因表达差异无显著性意义(P > 0.05)。诱导液中加入脂多糖刺激后，Alp、Ocn、Runx2表达均显著减少，差异有显著性意义。成骨诱导2周后，ob/ob小鼠骨髓间充质干细胞中Ocn、Runx2基因表达均低于C57小鼠骨髓间充质干细胞，Nf-κb在ob/ob小鼠骨髓间充质干细胞中表达略高，但差异无显著性意义。诱导液中加入脂多糖刺激后，Ocn、Runx2基因在两种小鼠骨髓间充质干细胞中表达均显著减少，差异有显著性意义(P < 0.05)，且在ob/ob小鼠骨髓间充质干细胞中表达显著低于C57小鼠骨髓间充质干细胞，差异有显著性意义(P < 0.05)。诱导2周时Ocn表达比诱导1周显著增加，差异有显著性意义(P < 0.05)。诱导2周时Runx2表达比诱导1周略降低，但差异无显著性意义(P > 0.05)，见图6-8。"

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