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中华关节外科杂志(电子版) ›› 2021, Vol. 15 ›› Issue (04) : 432 -437. doi: 10.3877/cma.j.issn.1674-134X.2021.04.007

基础论著

异鼠李素激活p38信号促进鼠间充质干细胞成骨分化
凡军1,†(), 曹丽萍1   
  1. 1. 201599　上海市金山区亭林医院骨科
  • 收稿日期:2020-04-22 出版日期:2021-09-29
  • 通信作者: 凡军

Isorhamnetin activates p38 signaling to promote osteogenic differentiation of murine mesenchymal stem cells

Jun Fan1,†(), Liping Cao1   

  1. 1. Department of Orthopedics, Tinglin Hospital, Jinshan District, Shanghai 201599, China
  • Received:2020-04-22 Published:2021-09-29
  • Corresponding author: Jun Fan
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凡军, 曹丽萍. 异鼠李素激活p38信号促进鼠间充质干细胞成骨分化[J]. 中华关节外科杂志(电子版), 2021, 15(04): 432-437.

Jun Fan, Liping Cao. Isorhamnetin activates p38 signaling to promote osteogenic differentiation of murine mesenchymal stem cells[J]. Chinese Journal of Joint Surgery(Electronic Edition), 2021, 15(04): 432-437.

目的

探究异鼠李素对骨髓间充质干细胞成骨分化的影响。

方法

3周龄C57BL/6J小鼠骨髓间充质干细胞在成骨诱导液下诱导成骨分化。细胞计数试剂盒(CCK-8)检测不同浓度的异鼠李素对成骨细胞毒性影响并行t检验以分析最低有效浓度。碱性磷酸酶(ALP)及茜素红染色观察有效浓度的异鼠李素对骨髓间充质干细胞成骨分化及矿化的影响。通过RT-PCR技术检测异鼠李素促成骨细胞分化的相关基因，包括骨桥蛋白(OPN)、骨钙蛋白(OCN)和ALP，并行t检验分析。蛋白印迹及免疫荧光技术验证异鼠李素促成骨分化相关通路及蛋白表达。

结果

5 μmol/L(t＝5.4，P<0.01)和10 μmol/L(t＝7.2，P<0.01)异鼠李素对骨髓间充质干细胞增殖有促进作用，并且呈现浓度依赖性，而20 μmol/L(t＝4.2，P<0.01)异鼠李素明显抑制了细胞增殖，有毒副作用。通过碱性磷酸酶及茜素红染色染色发现10 μmol/L异鼠李素碱性治疗的碱性磷酸酶染色及形成的钙结节较多。通过蛋白印迹技术发现，异鼠李素激活了p38信号通路。

结论

10 μmol/L异鼠李素通过激活p38信号通路达到促进骨髓间充质干细胞成骨分化。

关键词: 成骨细胞, 信号传导, 间质干细胞
Objective

To investigate the effects of isorhamnetin on osteogenic differentiation of bone marrow mesenchymal stem cells.

Methods

The bone marrow mesenchymal stem cells of three-week-old C57BL/6J mouse were induced to osteogenic differentiation under osteogenic-medium. The effects of isorhamnetin among different concentrations on osteoblast toxicity were detected by cell counting kit-8 reagent and analyzed by t test for the lowest effective concentration. Alkaline phosphatase (ALP) and alizarin red (AR) staining kits were used to observe the effects of isorhamnetin on osteogenic differentiation and mineralization at effective concentrations. Real time (RT)-PCR was used to detect the expression of osteoblast-related genes, including osteopontin (OPN), osteocalcin (OCN), and ALP, the data were analyzed by t test. Western blot and immunofluorescence were used to confirm that isorhamnetin promoted osteogenic differentiation-related pathways.

Results

The isorhamnetin concentrations of 5 μmol/L (t=5.4, P<0.01) and 10 μmol/L (t=7.2, P<0.01) promoted the proliferation of bone marrow mesenchymal stem cells, while isorhamnetin of 20 μmol/L (t=4.2, P<0.01) significantly inhibited cell proliferation and exhibited toxic effects. The ALP and AR staining revealed that 10 μmol/L isorhamnetin significantly promoted osteogenic differentiation and mineralization compared with the control group. It was found that isorhamnetin activated the p38 signaling pathway.

Conclusion

The isorhamnetin of 10 μmol/L promotes osteogenic differentiation of bone marrow mesenchymal stem cells by activating the p38 signaling pathway.

Key words: Osteoblast, Signal transduction, Mesenchymal stem cells
图1 不同浓度下ISO(异鼠李素)对细胞增殖的影响
图2 ISO(异鼠李素)对成骨细胞分化及矿化的影响。图A为空白组7 d后的ALP染色；图B为空白组21 d的AR染色；图C为实验组7 d的ALP染色；图D为实验组21 d后AR染色
图3 ISO(异鼠李素)对成骨细胞分化相关基因及蛋白的影响。图A~C示实验组中成骨分化相关基因表达比对照组明显增多；图D~G为WB(蛋白印迹法)结果，示实验组中成骨分化相关蛋白表达比对照组明显增多
图4 ISO(异鼠李素)激活p-p38(磷酸化p38)信号通路。图A~B为DAPI(4′，6-二脒基-2-苯基吲哚)染色，蓝色荧光显示细胞核；图C~D为p-p38染色，绿色荧光显示p-p38蛋白；图E为WB(蛋白印迹法)结果，示实验组中p-p38高表达；图F为siRNA干扰后WB(蛋白印迹法)结果，示实验组的p-p38蛋白表达减弱
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