Effect of recombinant Mycobacterium tuberculosis heat shock protein 10 on proliferation of human osteoblasts and regulation of bone metabolism

doi:10.3969/j.issn.2095-4344.2014.11.005

Abstract

Abstract:

BACKGROUND: Mycobacterium tuberculosis heat shock protein 10 (r-Mt cpn10) is one of the main factors that cause bone tuberculosis dissolution and absorption as well as inhibits the proliferation of osteoblasts. Receptor activator of nuclear factor kappa B ligand and osteoprotegerin are the important factors influencing bone metabolism.

OBJECTIVE: To observe the effect of r-Mt cpn10 on human osteoblast proliferation, alkaline phosphatase secretion, expression of receptor activator of nuclear factor-kappa B ligand mRNA and osteoprotegerin mRNA.

METHODS: Human bone marrow stromal cells were induced to differentiate into osteoblasts, and osteoblasts at passage 3 were cultured with various concentrations of r-Mt cpn10 (0.1, 1, 10 mg/L). Osteoblasts cultured without r-Mt CPN10 were assigned as controls.

RESULTS AND CONCLUSION: MTT assay results showed that, compared with control group, r-Mt cpn10 at different concentrations inhibited osteoblast proliferation and alkaline phosphatase secretion (P < 0.05). RT-PCR analysis showed that, r-Mt cpn10 at different concentrations increased receptor activator of nuclear factor-kappa B ligand mRNA expression (P < 0.01), and inhibited osteoprotegerin mRNA expression in a concentration-dependent manner (P < 0.01). 10 mg/L r-Mt cpn10 exhibited the strongest effect (P < 0.01). The r-Mt cpn10 can inhibit osteoblast proliferation and alkaline phosphatase activity, and it may influence bone metabolism by regulating the expression of receptor activator of nuclear factor-kappa B ligand mRNA and osteoprotegerin mRNA.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: osteoblasts, cell proliferation, heat-shock proteins, alkaline phosphatase

CLC Number:

R318

Zhang Yuan-yu, Liu Xia, Li Kun, Guo Yong-rong, Bai Jing-ping. Effect of recombinant Mycobacterium tuberculosis heat shock protein 10 on proliferation of human osteoblasts and regulation of bone metabolism[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(11): 1665-1671.

Figures/Tables 2

2.1 体外培养的人成骨细胞的形态倒置显微镜每日观察，刚接种的成骨细胞呈球形，细胞悬液于培养液中并逐渐沉降贴壁；培养24 h后，存活细胞已完全贴壁展开，此时细胞形态不规则，多呈三角形，多角形，有较多突起，单核呈卵圆形，1-3 个核仁，胞质丰富、胞浆清晰；传代后的细胞增殖迅速，培养三四小时，细胞开始贴壁，培养6-8 h贴壁基本完全，第3天细胞数量明显增多，培养五六天可长满瓶底，融合成片状，细胞排列更整齐、分布更均匀、生长更匀一，杂细胞已较少。原代培养约12 d细胞可长满瓶底。培养7-21 d生长期细胞分离相多见，细胞突起互相连接，汇合时，细胞呈铺路石状，并可重叠生长，重叠生长的细胞逐渐形成细胞小结，随后胶原堆积及钙盐沉积，形成不透光矿化结节(图2)。 2.2 重组结核杆菌热休克蛋白10对人成骨细胞增殖的影响 MTT实验检测显示，对照组成骨细胞吸光度随时间延长而逐渐升高，与对照组相比，0.1，1，10 mg/L重组结核杆菌热休克蛋白10组的成骨细胞吸光度随时间延长而逐渐降低，都呈时间依赖性抑制成骨细胞增殖，其中1，10 mg/L组72 h对成骨细胞增殖有抑制作用(P < 0.05)，其中 10 mg/L重组结核杆菌热休克蛋白10组的抑制作用最明显(P < 0.05)，见表1。 2.3 重组结核杆菌热休克蛋白10对人成骨细胞后碱性磷酸酶活性的影响成骨细胞培养在1，3，5，7，9 d 后，进行碱性磷酸酶活性测定。结果显示，细胞碱性磷酸酶活性随时间的延长逐渐下降，其中第1天和第5天的碱性磷酸酶活性与其他时间点相比较高，在培养第1天和第5天2个时间点上，1，10 mg/L重组结核杆菌热休克蛋白10组与对照组比较碱性磷酸酶活性明显下降(P < 0.05)，见表2。 2.4 成骨细胞的总mRNA的浓度检测和质量鉴定重组结核杆菌热休克蛋白10干预72 h后，提取各浓度干预下成骨细胞的总mRNA，经用核酸蛋白分析仪测定A260 nm/A280 nm，所有的样品的A260 nm/A280 nm均在1.7-2.0之间，提取各样品总RNA 3 μL，经10 g/L琼脂糖凝胶电泳分析，电泳结束后，在紫外灯下观查有两条带，28S和18S核糖体RNA条带非常亮而浓，没有出现因DNA污染而出现的弥散迁移带，提示未有降解和污染。 2.5 重组结核杆菌热休克蛋白10对成骨细胞护骨素、核因子κB受体活化因子配体mRNA表达的影响重组结核杆菌热休克蛋白10干预人成骨细胞72 h，可剂量依赖性增加核因子κB受体活化因子配体mRNA表达，质量浓度10 mg/L 重组结核杆菌热休克蛋白10作用最大，约为对照组表达量的322.7倍(P < 0.01)。重组结核杆菌热休克蛋白10浓度依赖性降低护骨素mRNA表达，干预72 h，质量浓度10 mg/L 重组结核杆菌热休克蛋白10降低作用明显(P < 0.01)。骨保护素/核因子κB受体活化因子配体值动态地反应成骨或破骨两种相反的趋势，质量浓度10 mg/L重组结核杆菌热休克蛋白10的骨保护素/核因子κB受体活化因子配体比值 0.001 9，相比较对照组1.07明显降低，显示倾向于破骨效应的趋势(表3)。"

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