›› 2012, Vol. 32 ›› Issue (3): 274-.doi: 10.3969/j.issn.1674-8115.2012.03.008

• 论著(基础研究) • 上一篇    下一篇

低氧/低氧诱导因子-1α通路对成骨细胞与破骨细胞耦联的调控作用

王络文, 邓廉夫, 朱雅萍, 魏 立, 齐 进   

  1. 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 200025
  • 出版日期:2012-03-28 发布日期:2012-03-28
  • 通讯作者: 邓廉夫, 电子信箱: lfdeng@msn.com。
  • 作者简介:王络文(1980—), 男, 硕士生;电子信箱: wenwen_1103@126.com。
  • 基金资助:

    国家自然科学基金(30872641)

Regulation of interaction of osteoblasts and osteoclasts by hypoxia/hypoxia-inducible factor-1&alpha|pathway

WANG Luo-wen, DENG Lian-fu, ZHU Ya-ping, WEI Li, QI Jin   

  1. Shanghai Institute for Orthopedics, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
  • Online:2012-03-28 Published:2012-03-28
  • Supported by:

    National Natural Science Foundation of China, 30872641

摘要:

目的 探讨低氧/低氧诱导因子(HIF)-1α通路对成骨细胞与破骨细胞耦联的调控作用。方法 取出生2~3 d条件性基因敲除小鼠颅盖骨的成骨细胞与4~8周龄C57BL/6小鼠股骨破骨细胞的前体细胞,建立基因敲除小鼠成骨细胞与破骨细胞前体细胞共培养体系(野生型、HIF-1α-/-、Vhl-/-、HIF-1α-/-/Vhl-/-共培养)。采用RT-PCR技术检测成骨细胞中核因子κB受体活化因子配体(RANKL)mRNA和骨保护素(OPG)mRNA的表达以及破骨细胞中标志酶基因TRAP mRNA的表达,甲苯胺蓝染色观察破骨细胞溶骨形成的骨陷窝。结果 RT-PCR检测结果显示:与野生型共培养比较,HIF-1α-/-共培养的成骨细胞中RANKL mRNA表达上调、OPG mRNA表达下调(均P<0.05),破骨细胞TRAP mRNA表达上调;Vhl-/-共培养和HIF-1α-/-/Vhl-/-共培养的成骨细胞中RANKL mRNA表达下调、OPG mRNA表达显著上调(均P<0.05),破骨细胞TRAP mRNA表达下调;随着共培养时间的延长,成骨细胞中RANKL mRNA和OPG mRNA表达均逐渐减少,而破骨细胞TRAP mRNA表达均逐渐增加。甲苯胺蓝染色倒置显微镜观察显示:共培养第9天,破骨细胞骨吸收陷窝出现;随着共培养时间的延长,骨吸收陷窝面积和深度逐渐增加;与野生型共培养比较,HIF-1α-/-共培养的破骨细胞骨吸收陷窝较大且较深,Vhl-/-和HIF-1α-/-/Vhl-/-共培养的破骨细胞骨吸收陷窝较小且较浅。结论 低氧/HIF-1α通路激活后,成骨细胞抑制破骨细胞的分化功能;低氧/HIF-1α通路阻断后,成骨细胞促进破骨细胞的分化功能。

关键词: 成骨细胞, 破骨细胞, 共培养, 低氧诱导因子-1α, Vhl, 基因敲除

Abstract:

Objective To investigate the regulation of interaction of osteoblasts and osteoclasts by hypoxia/hypoxia-inducible factor-1α (HIF-1α) pathway. Methods Osteoblasts were obtained from newborn (2 to 3 d) conditional gene knockout mice, osteoclast precursor cells were harvested from C57BL/6 mice aged 4 to 8 weeks, and co-culture system of osteoblasts and osteoclast precursor cells was established (wild-type, HIF-1α-/-, Vhl-/-, HIF-1α-/-/Vhl-/- co-culture). RT-PCR was employed to detect the expression of receptor activator of nuclear factor-κB ligand (RANKL) mRNA and osteoprotegerin (OPG) mRNA in osteoblasts and that of marker enzyme gene TRAP mRNA in osteoclasts, and resorption pits produced by osteoclasts were observed with toluidine blue staining. Results RT-PCR revealed that compared with wild-type co-culture, the expression of RANKL mRNA increased and that of OPG mRNA decreased in osteoblasts of HIF-1α-/- co-culture (P<0.05 for both), the expression of TRAP mRNA increased in osteoclasts of HIF-1α-/- co-culture, the expression of RANKL mRNA decreased and that of OPG mRNA increased in osteoblasts of Vhl-/-co-culture and HIF-1α-/-/Vhl-/- co-culture (P<0.05 for all), and the expression of TRAP mRNA decreased in osteoclasts of Vhl-/-co-culture and HIF-1α-/-/Vhl-/- co-culture. With the time increase of co-culture, the expression of RANKL mRNA and OPG mRNA in osteoblasts gradually decreased, and that of TRAP mRNA in osteoclasts gradually increased. Observation under inverted microscope with toluidine blue staining indicated that resorption pits of osteoclasts began to appear on the ninth day of co-culture, the area and depth of resorption pits gradually increased with the time increase of co-culture, the area and depth of resorption pits increased after HIF-1α-/- co-culture, and those decreased after Vhl-/- co-culture and HIF-1α-/-/Vhl-/- co-culture. Conclusion Osteoblasts may inhibit the differentiation of osteoclasts after the activation of hypoxia/HIF-1α pathway, while osteoblasts may promote the differentiation of osteoclasts after interruption of hypoxia/HIF-1α pathway.

Key words: osteoblast, osteoclast, co-culture, hypoxia-inducible factor-1α, Vhl, gene knockout