矮壮素对小鼠前成骨细胞系MC3T3-E1骨骼发育相关基因及蛋白的影响及其机制

doi:10.3969/j.issn.1004-616x.2018.03.005

癌变·畸变·突变 ›› 2018, Vol. 30 ›› Issue (3): 188-193,199.doi: 10.3969/j.issn.1004-616x.2018.03.005

矮壮素对小鼠前成骨细胞系MC3T3-E1骨骼发育相关基因及蛋白的影响及其机制

贾丽霞, 张琪, 侯晓红, 孟庆贺, 黄尧, 周文娟, 郝卫东

北京大学公共卫生学院食品安全毒理学研究与评价北京市重点实验室, 北京 100191

收稿日期:2018-02-20 修回日期:2018-05-02 出版日期:2018-05-30 发布日期:2018-05-30
通讯作者: 郝卫东,E-mail:whao@bjmu.edu.cn E-mail:whao@bjmu.edu.cn
作者简介:贾丽霞,E-mail:18811333069@163.com。
基金资助:
国家自然科学基金（81473002，81773446）

Effects of chlorocholine chloride on expression of skeletal development-related genes and proteins in MC3T3-E1 cells

JIA Lixia, ZHANG Qi, HOU Xiaohong, MENG Qinghe, HUANG Yao, ZHOU Wenjuan, HAO Weidong

Beijing Key Laboratory of Toxicological Research and Risk Assessment, School of Public Health, Peking University, Beijing 100191, China

Received:2018-02-20 Revised:2018-05-02 Online:2018-05-30 Published:2018-05-30

摘要/Abstract

摘要： 目的：探讨矮壮素（chlorocholine chloride，CCC）对小鼠前成骨细胞系MC3T3-E1细胞骨骼发育相关基因及蛋白表达情况的影响，评价矮壮素对骨骼发育的影响，并探究其可能的作用机制。方法：选取处于对数生长期的MC3T3-E1细胞接种于96孔板中，以8、40、200、1 000 μg/mL矮壮素进行染毒。MTT法检测矮壮素染毒后不同时间（24、48、72 h）MC3T3-E1细胞的相对存活率。实时荧光定量PCR法检测矮壮素染毒48 h后MC3T3-E1细胞中骨骼发育相关基因mRNA的表达情况，包括成骨相关基因碱性磷酸酶（ALP）、骨钙素（OCN）、胰岛素样生长因子1（IGF-1）、生长激素受体（GHR）；破骨相关基因核因子κB受体活化因子配体（RANKL）、骨保护素（OPG）、巨噬细胞集落刺激因子（M-CSF）。使用Western blot测定矮壮素染毒48 h后MC3T3-E1细胞中骨骼发育相关功能蛋白及MAPK信号通路蛋白的表达情况，包括碱性磷酸酶（ALP）、生长激素受体（GHR）、骨形成蛋白2（BMP2）、转录因子Runt相关蛋白2（Runx2）。结果：不同浓度矮壮素对MC3T3-E1细胞染毒不同时间均无明显细胞毒性；矮壮素染毒24 h，1 000 μg/mL矮壮素染毒组细胞中ALP、OCN、RANKL mRNA表达明显增高（P < 0.05），且细胞中RANKL与OPG的mRNA表达量比值明显上升（P < 0.05），GHR、IGF-1、OPG、M-CSF mRNA的表达无明显变化（P > 0.05）。1 000 μg/mL组MC3T3-E1细胞GHR和BMP2蛋白表达明显降低，磷酸化ERK蛋白表达量降低和磷酸化JNK蛋白表达量升高。结论：矮壮素对MC3T3-E1细胞无明显细胞毒性，但可能会抑制MC3T3-E1细胞向成熟成骨细胞的分化，且有可能促进破骨细胞分化；其作用机制可能与MAPK通路有关，磷酸化ERK降低和磷酸化JNK升高可能会抑制小鼠前成骨细胞骨骼发育相关蛋白的表达。

关键词: 矮壮素, 成骨细胞, 骨骼发育, MAPK通路

Abstract: OBJECTIVE: To investigate the effects of chlorocholine chloride (CCC) on expression of skeletal development-related genes and functional proteins in mouse pre-osteoblast cell line MC3T3-E1. METHODS: MC3T3-E1 cells in logarithmic growth phase were treated with 0,8,40,200,1 000 μg/mL CCC. At different time points (24,48,72 h) after treatment,cytotoxicity was determined by using the MTT assay. At 48 h after treatment,mRNA expression of skeletal and osteogenic development-related genes was detected using qPCR:alkaline phosphatase (ALP),osteocalcin(OCN),growth factor 1 (IGF-1),growth hormone receptor (GHR),RANKL,osteoprotegerin (OPG),macrophage colony-stimulating factor (M-CSF). In addition,expression of functional proteins related to bone development and MAPK signal pathways was determined by Western blot:ALP,GHR,bone morphogenetic protein 2 (BMP2),transcription factor runt-related protein 2 (Runx2). RESULTS: CCC had no significant effect on cytotoxicity of MC3T3-E1 at different time points (24,48,72 h). mRNA expression of ALP,OCN and RANKL in the 1 000 μg/mL groups increased significantly (P < 0.05);the ratio of RANKL and OPG in the 1 000 μg/mL group increased significantly (P < 0.05);mRNA expression of GHR,IGF-1,OPG,M-CSF did not change significantly (P > 0.05). Protein expression of GHR and BMP2 in the 1 000 μg/mL group was significantly decreased,while ALP and RUNX2 did not change significantly. Phosphorylated ERK protein expression decreased and phosphorylated JNK protein expression increased. CONCLUSION: CCC has almost no cytotoxicity on pre-cultured mouse MC3T3-E1 cells,but inhibited the differentiation of MC3T3-E1 cells into mature osteoblasts and promoted osteoclast differentiation. Moreover,involvement of mitochondria on differentiation of pre-mouse osteoblasts might be related to the MAPK pathway. The decrease of phosphorylated ERK and the increase of phosphorylated JNK might have inhibited migration of MC3T3-E1 to mature osteoblasts differentiation of cells,hence their differentiation to become mature osteoblasts.

Key words: chlorocholine chloride, osteoblast, bone development, MAPK pathway

中图分类号:

R994.6

贾丽霞, 张琪, 侯晓红, 孟庆贺, 黄尧, 周文娟, 郝卫东. 矮壮素对小鼠前成骨细胞系MC3T3-E1骨骼发育相关基因及蛋白的影响及其机制[J]. 癌变·畸变·突变, 2018, 30(3): 188-193,199.

JIA Lixia, ZHANG Qi, HOU Xiaohong, MENG Qinghe, HUANG Yao, ZHOU Wenjuan, HAO Weidong. Effects of chlorocholine chloride on expression of skeletal development-related genes and proteins in MC3T3-E1 cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(3): 188-193,199.

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矮壮素对小鼠前成骨细胞系MC3T3-E1骨骼发育相关基因及蛋白的影响及其机制

Effects of chlorocholine chloride on expression of skeletal development-related genes and proteins in MC3T3-E1 cells

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