Chinese Journal of Tissue Engineering Research ›› 2019, Vol. 23 ›› Issue (25): 3978-3986.doi: 10.3969/j.issn.2095-4344.1776
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Revised:
2019-02-26
Online:
2019-09-08
Published:
2019-09-08
Contact:
Tian Xiaobin, Professor, Doctoral supervisor, Guizhou Medical University, Guiyang 550025, Guizhou Province, China;
Sun Li, MD, Chief physician, Doctoral supervisor, Department of Orthopedics, Guizhou Provincial People’s Hospital, Guiyang 550002, Guizhou Province, China
About author:
Li Xianghe, Master candidate, Guizhou Medical University, Guiyang 550025, Guizhou Province, China
Supported by:
the National Natural Science Foundation of China, No. 81560356 (to TXB); Social Development Project of Guizhou Province, No. (2015) 3044 (to SL)
CLC Number:
Li Xianghe, Luo Wei, Hu Junxian, Yang Jing, Han Xinyun, Dong Shiwu, Yang Xianteng, Li Senlei, Yan Zhihui, Nie Yingjie, Tian Xiaobin, Sun Li.
2.1 白杨素对RAW264.7细胞活性的影响 0,20,40,60,80 μg/L白杨素处理RAW264.7细胞24-72 h后,CCK8分析结果显示,白杨素质量浓度在80 μg/L时能明显抑制细胞增殖,而在质量浓度为20-60 μg/L时,细胞增殖不受影响,见图2。因此,后续研究中白杨素的质量浓度设为20和60 μg/L。 2.2 白杨素抑制RANKL诱导的破骨细胞分化和形成 用RANKL(50 μg/L)、巨噬细胞集落刺激因子(25 μg/L)和白杨素(0,20,60 μg/L)诱导RAW264.7细胞分化为破骨细胞。通过抗酒石酸酸性磷酸酶染色分析白杨素对RANKL诱导的破骨细胞分化的影响。结果显示,白杨素显著抑制RANKL诱导的破骨细胞生成,且以质量浓度依赖的方式抑制RAW264.7细胞中抗酒石酸酸性磷酸酶阳性多核细胞数量,见图3A,B。此外,通过FAK染色分析破骨细胞中F-actin环结构的形成,F-actin环结构对破骨细胞骨吸收功能是必不可少的[19]。结果显示,白杨素质量浓度为60 μg/L时,F-actin环结构的尺寸明显减小,见图3C-E。表明白杨素能有效抑制破骨细胞分化和形成。"
2.3 白杨素在体外降低骨吸收活性 由于白杨素抑制RANKL诱导的破骨细胞生成,实验利用骨陷窝形成分析白杨素是否能抑制破骨细胞体外骨吸收活性。结果显示,白杨素以浓度依赖的方式明显的抑制骨吸收面积,见图4。 2.4 白杨素抑制RANKL诱导的破骨细胞标志基因表达 为进一步阐明白杨素对破骨细胞分化的影响,实时定量PCR结果显示,在破骨细胞分化过程中,RANKL显著上调破骨细胞特异性基因NFATc1、C-FOS、组织蛋白酶K、抗酒石酸酸性磷酸酶、OC-STAMP、DC-STAMP的mRNA表达水平。而白杨素以浓度依赖的方式对上述mRNA表达具有抑制作用,见图5A-F。Western blot分析结果表明,RANKL明显上调了NFATc1和C-FOS的表达,而白杨素则抑制这2种蛋白的表达图5G。该结果与实时定量PCR结果一致。表明白杨素在体外可以抑制RANKL诱导的破骨细胞关键基因表达。 2.5 白杨素通过抑制核因子κB信号通路来抑制破骨细胞分化 RANKL诱导的核因子κB信号通路的激活对破骨细胞的分化和功能是必不可少的[20]。为了探讨白杨素在破骨细胞发生中的作用机制,在白杨素(60 μg/L)存在或不存在的条件下,用RANKL(50 μg/L)和巨噬细胞集落刺激因子 (25 μg/L)分别处理RAW264.7细胞0,15,30和60 min,Western blot分析结果显示,当白杨素(60 μg/L)存在时, 核因子κB p65的磷酸化表达被白杨素下调。实验进一步发现,白杨素同时还显著抑制IκBα的磷酸化和退化,见图5H-M。表明,白杨素通过减弱核因子κB信号通路的活性来抑制RANKL诱导的破骨细胞分化。"
2.6 白杨素下调脂多糖诱导的破骨细胞形成 为了研究白杨素在脂多糖介导的炎症条件下对破骨细胞分化的影响,RAW264.7细胞使用RANKL(50 μg/L)和巨噬细胞集落刺激因子(25 μg/L)预处理24 h。然后,这些细胞再用脂多糖(100 μg/L)和白杨素(0,20,60 μg/L)孵育72 h,诱导细胞分化为破骨细胞,抗酒石酸酸性磷酸酶染色用来分析脂多糖诱导的破骨细胞分化。作者观察到抗酒石酸酸性磷酸酶阳性多核破骨细胞呈浓度依赖性的减少,见图6A,B。 为了进一步研究白杨素对脂多糖诱导破骨细胞分化的影响,采用实时定量PCR方法对多种破骨细胞标志基因进行分析。如图6所示,白杨素明显下调这些标记基因的表达,与抗酒石酸酸性磷酸酶染色结果一致,见图6C-H。 同样,利用Western blot分析脂多糖诱导的NFATc1和C-FOS蛋白表达,也获得了相同的结果,NFATc1和C-FOS的蛋白表达均明显降低,见图6I,M,N。表明白杨素在脂多糖诱导的炎症条件下对破骨细胞的形成有负性调节作用。 因为白杨素抑制脂多糖诱导的破骨细胞形成,因此分析了在脂多糖刺激条件下,能够促进破骨细胞形成并导致骨丢失的促炎细胞因子(肿瘤坏死因子α、白细胞介素1β、白细胞介素6)的表达情况。实时定量PCR在mRNA水平上分析这些基因的表达。结果显示白杨素能显著抑制这些促炎细胞因子基因的表达,见图6J-L。"
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