黄精多糖对RANKL诱导骨髓巨噬细胞向破骨细胞分化及体内骨吸收功能的影响  

Polygonatum sibiricum polysaccharide attenuates bone marrow-derived macrophages to differentiate into osteoclasts and protects against lipopolysaccharide-induced osteolysis in vivo

作　　者：何基琛 宗少晖[1] 曾高峰[2] 杜力[1] 彭小明[1] 施雄智 吴云乐 

机构地区：[1]广西医科大学第一附属医院脊柱骨病外科,广西壮族自治区南宁市530021 [2]广西医科大学公共卫生学院,广西壮族自治区南宁市530021

出　　处：《中国组织工程研究》2017年第20期3117-3122,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(81360279)~~

摘　　要：背景:骨髓巨噬细胞具有向破骨细胞分化的潜能。黄精多糖可能抑制骨髓巨噬细胞向破骨细胞分化,有望成为治疗骨质疏松的新药物。目的:探讨黄精多糖对核因子κB受体活化因子配体(RANKL)诱导的小鼠骨髓巨噬细胞向破骨细胞分化及体内骨吸收的影响。方法:培养小鼠骨髓巨噬细胞,在巨噬细胞集落刺激因子和不同质量浓度黄精多糖(5,10,20,40,80,160,320,640,1 280,2 560 mg/L)干预下,CCK-8法检测黄精多糖对小鼠骨髓巨噬细胞增殖的影响以确定黄精多糖质量浓度范围;在巨噬细胞集落刺激因子和RANKL诱导下给予5,10,20,40,80,160,320,640 mg/L黄精多糖进行干预,不加黄精多糖作为对照组,显微镜下观察细胞形态变化。抗酒石酸酸性磷酸酶染色鉴定破骨细胞数目;实时荧光定量PCR检测破骨相关基因抗酒石酸酸性磷酸酶、金属基质蛋白酶9、组织蛋白酶K、活化T细胞核因子c1 mRNA表达。建立脂多糖诱导小鼠颅骨骨溶解模型并给予黄精多糖干预,采用Micro-CT扫描及相关软件定量分析骨体积/组织体积百分比、骨小梁数目、骨小梁间距、骨小梁厚度,并制备组织切片抗酒石酸酸性磷酸酶染色鉴定破骨细胞数目及定量分析骨吸收面积。结果与结论:(1)与对照组相比,质量浓度在640 mg/L以下的黄精多糖对小鼠骨髓巨噬细胞增殖无明显影响(P>0.05);(2)不同质量浓度的(40-640 mg/L)黄精多糖可不同程度的减少破骨细胞数目(P<0.01),显著降低了破骨细胞的分化成熟,同时明显降低抗酒石酸酸性磷酸酶、金属基质蛋白酶9、组织蛋白酶K、活化T细胞核因子c1基因的表达(P<0.05);(3)与脂多糖组相比,黄精多糖能有效缓解脂多糖诱导的颅骨骨溶解,骨体积/组织体积百分比、骨小梁数目、骨小梁间距均减小(P<0.05),破骨细胞数目及骨吸收面明显减少(P<0.01);(4)说明黄精多糖能够抑制小鼠骨髓巨噬细胞向破骨细胞分化成熟及�BACKGROUND： Bone marrow-derived mononuclear cells（BM-MNCs） hold the potential of differentiating into osteoclasts. Polygonatum sibiricum polysaccharide（PSP） may inhibit the differentiation of BM-MNCs into osteoclasts and it is expected to become a new drug for the treatment of osteoporosis. OBJECTIVE： To investigate the effect of PSP on the differentiation of mouse BM-MNCs into osteoclasts induced by receptor activator of nuclear factor kappa-B ligand（RANKL） and bone resorption in vivo. METHODS： Mouse bone marrow-derived macrophages cultured in vitro, the effect of macrophage colony stimulating factor and PSP（5, 10, 20, 40, 80,160, 320, 640, 1 280, 2 560 mg/L） on the proliferation of mouse BM-MNCs was detected by cell counting kit-8 assay to determine the PSP concentration range; the mouse BMMs were cultured and induced in DMEM medium containing macrophage colony stimulating factor, RANKL and 5, 10, 20, 40, 80,160, 320, 640 mg/L PSP, respectively; those cultured without PSP served as control group. The morphological changes of cells were observed under an inverted microscope.; the number of osteoclasts was detected by tartrate-resistant acid phosphatase staining; the mRNA expression levels of osteoclast-related genes including tartrate-resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, and nuclear factor of activated T cells c1 were evaluated by quantitative real-time PCR. A mouse model of calvarial osteolysis induced by lipopolysaccharide was established to receive PSP intervention, and then micro CT scanning, three-dimensional reconstruction and relevants software were used for quantitative analysis of bone volume/volume percentage, trabecular number, trabecular bone spacing and thickness. The number of osteoclasts was identified by tartrate-resistant acid phosphatase staining and quantitative analysis of bone resorption area was conducted. RESULTS AND CONCLUSION： Compared with the control group, the concentration of PSP below 640 mg/L showed no significant effect on th

关 键 词：黄精属 细胞分化 破骨细胞 骨质疏松 组织工程 组织构建 骨组织工程 黄精多糖 颅骨骨溶解模型 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]