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  • 孙斌,徐增,吴卉乔,郭蕾,张颖,石长贵,袁文.雷奈酸锶改善成骨不全症模型oim小鼠代谢失衡的双向机制[J].第二军医大学学报,2019,40(9):945-953    [点击复制]
  • SUN Bin,XU Zeng,WU Hui-qiao,GUO Lei,ZHANG Ying,SHI Chang-gui,YUAN Wen.Strontium ranelate improving metabolic imbalance in oim mice of osteogenesis imperfecta: the dual mechanism[J].Acad J Sec Mil Med Univ,2019,40(9):945-953   [点击复制]
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雷奈酸锶改善成骨不全症模型oim小鼠代谢失衡的双向机制
孙斌1,徐增1,吴卉乔1,郭蕾2,张颖1,石长贵1*,袁文1
0
(1. 海军军医大学(第二军医大学)长征医院脊柱外科, 上海 200003;
2. 上海交通大学医学院附属瑞金医院上海市伤骨科研究所, 上海市中西医结合防治骨与关节病损重点实验室, 上海 200025
*通信作者)
摘要:
目的 探讨雷奈酸锶对成骨不全症(OI)模型oim小鼠成骨细胞和破骨细胞的双重作用。方法 取1周龄OI模型纯合子oim/oim小鼠及野生型(wt/wt)小鼠的颅骨,采用Ⅰ型胶原酶通过连续消化法获取成骨细胞;取5~7周龄oim/oim小鼠和wt/wt小鼠的长骨,获取骨髓单核细胞诱导分化成破骨细胞。给予不同浓度(1、10 mmol/L)的雷奈酸锶进行干预,采用实时定量PCR(qRT-PCR)及蛋白质印迹法分别在mRNA及蛋白水平检测成骨细胞分化相关基因Runt相关转录因子2(Runx2)、碱性磷酸酶(ALP)、骨钙蛋白(OCN),破骨细胞分化相关基因降钙素受体(Calcr)、抗酒石酸磷酸酶(Trap)、组织蛋白酶K(CTSK),以及破骨分化相关转录因子c-fos、活化T细胞核因子c1(NFATc1)的表达;采用ALP染色及茜素红S染色观察成骨分化及矿化情况;采用Trap染色及骨片陷窝实验评估破骨细胞形成数量及骨吸收活性;采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)比色法检测雷奈酸锶对成骨细胞和破骨细胞活性的影响。结果 qRT-PCR及蛋白质印迹法检测结果显示,雷奈酸锶在mRNA及蛋白水平均可促进oim/oim小鼠成骨细胞分化相关基因(Runx2、ALP、OCN)的表达增加,同时抑制破骨细胞分化相关基因及转录因子(Calcr、Trap、CTSK、c-fos、NFATc1)的表达(P均<0.05),且随浓度增加作用增强。ALP染色及茜素红S染色结果显示,雷奈酸锶可促进oim/oim小鼠成骨细胞分化及矿化(P均<0.05)。Trap染色及骨片陷窝实验结果显示,雷奈酸锶可降低oim/oim小鼠破骨细胞形成数量和破骨细胞骨吸收活性(P均<0.05)。MTT结果显示,1 mmol/L、10 mmol/L雷奈酸锶对oim/oim小鼠成骨细胞和破骨细胞无细胞毒性。结论 雷奈酸锶可有效改善OI模型oim小鼠的骨代谢失衡,其机制可能是促进成骨细胞分化及矿化,同时抑制破骨细胞的形成及骨吸收活性。
关键词:  雷奈酸锶  成骨不全症  成骨细胞  破骨细胞  代谢
DOI:10.16781/j.0258-879x.2019.09.0945
投稿时间:2019-04-26修订日期:2019-06-05
基金项目:国家自然科学基金(81802120),上海市科学技术委员会科研计划项目(18YF1423100).
Strontium ranelate improving metabolic imbalance in oim mice of osteogenesis imperfecta: the dual mechanism
SUN Bin1,XU Zeng1,WU Hui-qiao1,GUO Lei2,ZHANG Ying1,SHI Chang-gui1*,YUAN Wen1
(1. Department of Spine Surgery, Changzheng Hospital, Naval Medical University(Second Military Medical University), Shanghai 200003, China;
2. Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases, Shanghai Institute of Traumatology and Orthopaedics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
*Corresponding author)
Abstract:
Objective To explore the dual effects of strontium ranelate (SrR) on primary osteoblasts and osteoclasts using an osteogenesis imperfecta (OI) model oim mouse. Methods The osteoblasts were obtained by digesting the calvaria of one-week-old homozygote oim/oim mice and wild type (wt/wt) mice with typeⅠcollagenase. The osteoclasts were obtained by inducing the bone marrow mononuclear cells derived from the long bone of oim/oim mice and wt/wt mice aged between five and seven weeks old. SrR with different concentrations (1 mmol/L and 10 mmol/L) was used to stimulate the cells. Quantitative real-time PCR (qRT-PCR) and Western blotting were used to quantify the expression of osteoblast differentiation-related genes (Runt-related transcription factor 2[Runx2], alkaline phosphatase[ALP] and osteocalcin[OCN]), osteoclast differentiation-related genes (calcitonin receptor[Calcr], tartrate-resistant acid phosphatase[Trap] and cathepsin K[CTSK]), and osteoclast differentiation-related transcription factors (cellular oncogene fos[c-fos] and nuclear factor of activated T-cell c1[NFATc1]). The osteoblast differentiation and mineralization were evaluated by ALP staining and alizarin red S staining. The osteoclastogenesis and resorption activity of the osteoclasts were evaluated by Trap staining and pit formation assay. The effect of SrR on cell viability of osteoblasts and osteoclasts was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results qRT-PCR and Western blotting showed that the expression levels of Runx2, ALP, and OCN in oim/oim mice were significantly increased at mRNA and protein levels after stimulation with SrR in a dose-dependent manner (all P<0.05). In comparison, SrR caused significant decreases in Calcr, Trap, CTSK, c-fos, and NFATc1 expression (all P<0.05). ALP staining and alizarin red S staining demonstrated that SrR stimulation significantly increased osteoblast differentiation and mineralization of oim/oim mice (all P<0.05). Trap staining and pit formation assay showed that SrR significantly decreased the formation number of osteoclasts and inhibited the resorption activity of osteoclasts in oim/oim mice (all P<0.05). MTT assay showed that 1 mmol/L and 10 mmol/L SrR had no cell cytotoxicity on osteoblasts or osteoclasts of oim/oim mice. Conclusion SrR can improve the imbalance of bone metabolism in OI model oim mouse, which might involve the increases in osteoblast differentiation and mineralization and decreases in formation and resorption of the osteoclasts.
Key words:  strontium ranelate  osteogenesis imperfecta  osteoblasts  osteoclasts  metabolism