中国组织工程研究

中国组织工程研究 ›› 2013, Vol. 17 ›› Issue (24): 4481-4487.doi: 10.3969/j.issn.2095-4344.2013.24.016

• 组织构建与生物活性因子 tissue construction and bioactive factors • 上一篇    下一篇

低氧诱导因子1α基因敲除小鼠椎间盘退变与益气化瘀方的干预

王 晶1, 2，董芳芳1, 2，李晓锋1, 2，许金海2，舒 冰1, 2，施 杞1, 2，王拥军1, 2，周重建1, 2   

  1. 1 上海中医药大学脊柱病研究所，上海市   200032
    2 上海中医药大学附属龙华医院，上海市   200032
  • 收稿日期:2012-10-13 修回日期:2012-12-06 出版日期:2013-06-11 发布日期:2013-06-11
  • 通讯作者: 周重建，博士，研究员，上海中医药大学脊柱病研究所，上海市 200032 zhouchongjian@hotmail.com
  • 作者简介:王晶★，女，1987年生，安徽省人，汉族，2010年上海中医药大学在读硕士，主要从事脊柱退变疾病研究。 188628850@qq.com
  • 基金资助:

    国家自然科学基金面上项目(30973760)；沪教委科[2009]6号，上海市高校创新团队计划

低氧诱导因子1α基因敲除小鼠椎间盘退变与益气化瘀方的干预

Wang Jing1, 2, Dong Fang-fang1, 2, Li Xiao-feng1, 2, Xu Jin-hai2, Shu Bing1, 2, Shi Qi1, 2, Wang Yong-jun1, 2, Zhou Chong-jian1, 2   

  1. 1 Institute of Spine Disease, Shanghai University of Traditional Chinese Medicine, Shanghai  200032, China
    2 Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai  200032, China
  • Received:2012-10-13 Revised:2012-12-06 Online:2013-06-11 Published:2013-06-11
  • Contact: Zhou Chong-jian, M.D., Investigator, Institute of Spine Disease, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China; Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China zhouchongjian@hotmail.com
  • About author:Wang Jing★, Studying for master’s degree, Institute of Spine Disease, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China; Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China 188628850@qq.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30973760*; the Foundation for Innovation Team Plan in Shanghai High Institutes, Shanghai Municipal Education Commission, No. [2009]6*

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摘要：

背景：低氧诱导因子1α与椎间盘软骨的正常生理及病理情况存在密切的关系，能够维持软骨组织在低氧环境中的正常活性，低氧诱导因子1α基因敲除后，软骨组织无法维持其正常低氧状态，导致软骨组织营养供应障碍，使软骨细胞处于异常缺血缺氧状态，长此以往，软骨组织会发生退变。
目的：观察低氧诱导因子1α条件性基因敲除小鼠椎间盘软骨退变情况，以及中药复方益气化瘀方对减缓低氧诱导因子1α基因敲除小鼠椎间盘软骨退变的作用。
方法：①将杂交繁殖获得同窝的低氧诱导因子1α+/+对照小鼠和低氧诱导因子1α基因敲除小鼠，分别分为为2.5月龄组和4.5月龄组(n=6)。分别在2.5、4.5月龄处死相应小鼠，取L4-6腰椎进行藏红固绿、苏木精-伊红染色及免疫组化相关指标染色及分析。②取12只0.5月龄低氧诱导因子1α基因敲除小鼠随机均分为生理盐水组和益气化瘀方组。灌胃给药2个月后，取两组小鼠的L4-6椎间盘进行藏红固绿、苏木精-伊红染色及免疫组化相关指标染色及分析。
结果与结论：①低氧诱导因子1α-/-小鼠：2.5月龄组椎间盘软骨组织出现破损和骨化，细胞分布不均匀，软骨细胞减少，椎间盘软骨中Ⅱ型胶原和Sox9表达降低，X型胶原、基质金属蛋白酶13表达增加；4.5月龄组椎间盘软骨损伤更加严重，Ⅱ型胶原蛋白与Sox9表达进一步降低， X型胶原、基质金属蛋白酶13表达进一步上升。②与生理盐水组比较，益气化瘀方干预后苏木精-伊红染色和藏红固绿染色显示软骨骨化和缺损减轻，软骨细胞数目较多，分布较均匀。与生理盐水组比较，益气化瘀方组椎间盘软骨中Ⅱ型胶原和Sox9的表达升高，X型胶原、基质金属蛋白酶13表达减少。说明低氧诱导因子1α基因敲除小鼠出现了椎间盘软骨退变，并且这种退变随着小鼠增龄而加重；益气化瘀方可以减轻低氧诱导因子1α基因敲除小鼠的椎间盘软骨退变。

关键词: 组织构建, 组织构建与生物活性因子, 益气化瘀方, 低氧诱导因子1α, 基因敲除小鼠, 椎间盘, 软骨, 退变, 骨化, 骨缺损, Ⅱ型胶原, 软骨细胞, 基质金属蛋白酶, 国家自然科学基金

Abstract:

BACKGROUND: Hypoxia-inducible factor 1 α is highly correlated with normal physiology and pathology of intervertebral disc cartilage because it can maintain normal activities of cartilage in hypoxia. After knockout of hypoxia-inducible factor 1α, cartilage cannot maintain the hypoxia state, resulting in nutritional disturbance, and hypoxia/ischemia in chondrocytes. Consequently, cartilage degeneration occurs.
OBJECTIVE: To observe the degeneration of intervertebral disc cartilage end-plate in hypoxia-inducible factor 1α gene knockout mice and investigate the effects of Chinese herbal compound Yiqihuayu Prescription on intervertebral disc cartilage end-plate degeneration.
METHODS: We collected 2.5- (n=6) and 4.5-month-old (n=6) hypoxia-inducible factor 1α gene knockout mice and wild type hypoxia-inducible factor 1α+/+ control mice were obtained by interbreeding. The mice were sacrificed at 2.5 and 4.5 months old. Lumbar vertebra at L4-6 levels was harvested for Safranin O/fast green, hematoxylin-eosin, immunohistochemical staining. Another 12 hypoxia-inducible factor 1α gene knockout mice of 0.5 month old were randomly assigned to normal saline and Yiqihuayu Prescription groups. Following intragastrical administration for 2 months, lumbar vertebra at L4-6 levels was harvested for Safranin O/fast green, hematoxylin-eosin, immunohistochemical staining and analysis.
RESULTS AND CONCLUSION: For hypoxia-inducible factor 1α-/- mice, mice at 2.5 months old developed aging related cartilage loss and bony tissue appearance, in addition to cartilage defects, uneven distribution of cells, and chondrocyte reduction. In addition, type II collagen and Sox-9 expression in intervertebral disc cartilage decreased, while type X collagen and matrix metalloproteinase 13 expression increased. At 4.5 months old, the cartilage injury was worsened, type II collagen and Sox-9 expression further reduced, and type X collagen and matrix metalloproteinase 13 expression further increased. Compared with normal saline group, Yiqihuayu Prescription reduced ossification and defect of endplate cartilage, the number of chondrocytes increased, and the distribution was more uniform as shown by Safranin O/fast green and hematoxylin-eosin staining. Moreover, Yiqihuayu Prescription increased type II collagen and Sox-9 expression, but decreased type X collagen and matrix metalloproteinase 13 protein expression. The results indicate that intervertebral disc cartilage degeneration occurred in hypoxia-inducible factor 1α gene knockout mice, which was progressed with aging of mice. Yiqihuayu Prescription can attenuate the degeneration of intervertebral disc cartilage in hypoxia-inducible factor 1α gene knockout mice.

Key words: tissue construction, tissue construction and bioactive factors, Yiqihuayu Prescription, hypoxia-inducible factor 1α, gene knockout mice, intervertebral disc, cartilage, degeneration, ossification, bone defect, type II collagen, chondrocyte, matrix metalloproteinase, National Natural Science Foundation of China

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