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浙江大学学报（医学版）
浙江大学学报（医学版）  2015, Vol. 44 Issue (3): 301-307    DOI: 10.3785/j.issn.1008-9292.2015.05.10
原著     
淫羊藿苷促进胶原水凝胶三维立体培养成骨细胞的成熟分化
谢艳芳1, 王鸣刚1, 陈克明2, 石文贵1, 周建2, 高玉海2
1. 兰州理工大学生命科学与工程学院, 甘肃 兰州 730050;
2. 兰州军区兰州总医院骨科研究所, 甘肃 兰州 730050
Icariin enhances differentiation and maturation of rat calvarial osteoblasts in collagen hydrogel three-dimensional culture
XIE Yan-fang1, WANG Ming-gang1, CHEN Ke-ming2, SHI Wen-gui1, ZHOU Jian2, GAO Yu-hai2
1. School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China;
2. Institute of Orthopedics, Lanzhou CPLC Command General Hospital, Lanzhou 730050, China
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摘要：

目的:研究淫羊藿苷对胶原水凝胶三维立体培养的新生大鼠颅骨成骨细胞(ROB)分化和成熟的影响。方法:取新生SD 大鼠颅骨,酶消化法培养ROB,将传代1次的ROB包埋于2 mg/mL的Ⅰ型鼠尾胶原中进行三维立体培养,48 h后进行双乙酸荧光素/碘化丙啶(FDA/PI)细胞染色、苏木素-伊红(HE)染色,并用扫描电镜观察细胞生长状态。分别采用终浓度为1×10-4、1×10-5、1×10-6和1×10-7 mol/L的淫羊藿苷进行干预,培养3、6、9 d后分别检测细胞内碱性磷酸酶活性,筛选最佳药物浓度;以最佳浓度淫羊藿苷干预三维凝胶中的ROB 12、24、36、48 h后,用实时定量PCR和蛋白质印迹法分别检测骨形态发生蛋白2(BMP-2)、Runt相关转录因子2(RUNX-2)、Osterix mRNA及其蛋白表达情况。结果:胶原水凝胶三维立体培养的ROB 采用FDA/PI染色、HE染色以及扫描电镜结构显示,ROB在胶原水凝胶中分布均匀,生长状态良好。终浓度为1×10-5、1×10-6、1×10-7 mol/L的淫羊藿苷可显著提高三维凝胶中ROB的碱性磷酸酶活性,其中以1×10-6 mol/L 浓度淫羊藿苷干预后ROB的碱性磷酸酶活性最高。1×10-6 mol/L 淫羊藿苷能显著提高BMP-2、RUNX-2、Osterix mRNA及其蛋白表达量。结论:淫羊藿苷可显著促进三维立体培养的ROB成骨性相关因子的表达,提示淫羊藿苷对胶原水凝胶三维立体培养的ROB具有较强的促骨形成活性。
关键词： 细胞外基质;  淫羊藿/投药和剂量;  淫羊藿甙;  水凝胶;  胶原;  成骨细胞/药物作用;  细胞;  培养的    
Abstract:

Objective: To investigate the effects of icariin on the differentiation and maturation of rat calvarial osteoblasts(ROB) in collagen hydrogel three-dimensional culture. Methods: ROB were obtained by enzyme digestion from the segregated neonatal SD rats skull and were embedded in 2 mg/mL rat tail collagen for three-dimensional culture. The growth state of ROB was observed by FDA/PI staining, HE staining and scanning electron microscopy. ROB were treated with icariin at the concentration of 1×10-4, 1×10-5, 1×10-6 and 1×10-7 mol/L respectively. The activity of alkaline phosphatase(ALP) was detected after 3, 6, 9 d of icariin treatment. Three-dimensional cultured ROB were treated with optimal concentration icariin for 12, 24, 36, 48 h and total RNA was extracted and the mRNA expressions of bone morphogenetic protein-2(BMP-2), Runt-related transcription factor 2(RUNX-2) and Osterix were detected by real time RT-PCR. The protein expression of BMP-2, RUNX-2 and Osterix were examined by Western-blotting. Results: ROB were cultured in collagen hydrogel successfully. FDA/PI staining, HE staining, and scanning electron microscopy showed that ROB adhered with collagen tightly and distributed homogeneously. Icariin at final concentration of 1×10-5, 1×10-6 and 1×10-7 mol/L all enhanced the activity of ALP of collagen hydrogel three-dimensional cultured ROB, and 1×10-6 mol/L was the optimal concentration. Besides,icariin(1×10-6 mol/L) increased mRNA and protein expression of BMP-2、RUNX-2 and Osterix compared to control group. Conclusion: Icariin can enhance the expression of osteogenic markers of ROB in collagen hydrogel three-dimensional culture significantly.
Key words: Extracellular matrix    Epimedium brevicornum/administration &    dosage    Icariin    Hydrogel    Collagen    Osteoblasts/drug effects    Cells, cultured
收稿日期: 2015-02-17 出版日期: 2015-05-25
:  Q233  
基金资助:

国家自然科学基金 (81270963,81471090)
通讯作者: 王鸣刚(1962-),男,博士,教授,硕士生导师,主要从事骨质疏松症的预防与治疗的研究;E-mail:mgwang@163.com;http://orcid.org/0000-0002-4222-6978     E-mail: mgwang@163.com
作者简介: 谢艳芳(1990-),女,硕士研究生,主要从事天然产物对骨质疏松症的预防与治疗作用研究;E-mail:xieyanfang1990@126.com;http://orcid.org/0000-0002-2978-7906
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引用本文:

谢艳芳, 王鸣刚, 陈克明, 石文贵, 周建, 高玉海. 淫羊藿苷促进胶原水凝胶三维立体培养成骨细胞的成熟分化[J]. 浙江大学学报（医学版）, 2015, 44(3): 301-307.

XIE Yan-fang, WANG Ming-gang, CHEN Ke-ming, SHI Wen-gui, ZHOU Jian, GAO Yu-hai. Icariin enhances differentiation and maturation of rat calvarial osteoblasts in collagen hydrogel three-dimensional culture. Journal of ZheJiang University(Medical Science), 2015, 44(3): 301-307.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2015.05.10    或    http://www.zjujournals.com/med/CN/Y2015/V44/I3/301

[1] SHEN P,WONG S P,LI J,et al. Simple and sensitive liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of five epimedium prenylflavonoids in rat sera[J]. J Chromatogr B Analyt Technol Biomed Life Sci,2009,877(1-2):71-78.
[2] 刘海江,王小平,林 娟,等. 淫羊藿苷和黄芪苷I对骨髓基质细胞增殖及成骨能力的影响[J].中药材,2006,29(10):1062-1065. LIU Hai-jiang,WANG Xiao-ping,LIN Juan,et al. The effect of Icariin and Astragalosid on the proliferation and differentiation of bone marrow stromal cells [J]. Chinese Herbal Medicines,2006,29(10):1062-1065. (in Chinese)
[3] BAJAJ P,SCHWELLER R M,KHADEMHOSSEINI A,et al. 3D biofabrication strategies for tissue engineering and regenerative medicine [J]. Annu Rev Biomed Eng,2014,16(23):247-276.
[4] 王 敏,兰 亚,胡 皓,等.以胶原水凝胶为支架构建干细胞三维立体培养系统[J]. 中国组织工程研究,2013,17(29):5323-5330. WANG Min,LAN Ya,HU Hao,et al. In vitro establishment of a three-dimensional hepatocyte culture system using collagen hydrogel as scaffolds [J]. Chinese Journal of Tissue Engineering Research,2013,17(29):5323-5330. (in Chinese)
[5] VERHEIJ J G,GERAETS W G,VAN DER STELT P F,et al. Prediction of osteoporosis with dental radiographs and age[J]. Dentomaxillofac Radiol,2009,38(7):431-437.
[6] KIM K,SVEDBOM A,LUO X,et al. Comparative cost-effectiveness of bazedoxifene and raloxifene in the treatment of postmenopausal osteoporosis in Europe[J]. Osteoporos Int,2014,25(1):325-337.
[7] BORRELLI F, ERNST E. Alternative and complementary therapies for the menopause[J]. Maturitas,2010,66(4):333-343.
[8] DRAKE M T,CLARKE B L,KHOSLA S,et al. Bisphosphonates: mechanism of action and role in clinical practice[J]. Mayo Clin Proc,2008,83(9):1032-1045.
[9] ZHOU J,MING L G,GE B F,et al. Effects of 50 Hz sinusoidal electromagnetic fields of different intensities on proliferation, differentiation and mineralization potentials of rat osteoblasts[J].Bone,2011,49(4):753-761.
[10] CHEN K M,GE B F,LIU X Y,et al. Icariin inhibits the osteoclast formation induced by RANKL and macrophage-colony stimulating factor in mouse bone marrow culture[J]. Pharmazie, 2007,62(5):388-391.
[11] 徐道志,詹红生,赵咏芳. 成骨细胞分化与骨骼发育的转录因子CbfA1/Runx2 [J].中医正骨, 2006, 18(11):61-63. XU Dao-zhi,ZHAN Hong-sheng,ZHAO Yong-fang. Transcription factors CbfA1/Runx2 of the differentiation of osteoblast and bone formation [J]. The Journal of Traditional Chinese Orthopedics and Traumatology,2006,18(11):61-63. (in Chinese)
[12] 余向前,张 柳. Osterix对成骨细胞分化影响的研究进展[J].华北煤炭医学院学报,2007,9(1):44-45. XU Xiang-qian, ZHANG Liu. The effect of Osterix on the differentiation of osteoblast [J]. Journal of North China Coal Medical College,2007,9(1):44-45. (in Chinese)
[13] 孙 强,邱 勇. 转录因子Runx-2, Osterix与骨髓间质干细胞的成骨分化[J].江苏医药, 2006,32(7):657-659. SUN Qiang,QIU Yong. The effect of Runx-2,Osterix in osteogenic differentiation process of marrow stromal cells [J]. Jiangsu Medical Journal,2006,32(7):657-659.(in Chinese)
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