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  

Intervertebral disc degeneration in hypoxia-inducible factor 1 alpha-knockout mice and treatment of Yiqihuayu Prescription

作　　者：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] 

机构地区：[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

出　　处：《中国组织工程研究》2013年第24期4481-4487,共7页Chinese Journal of Tissue Engineering Research

基　　金：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~~

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�BACKGROUND： Hypoxia-inducible factor 1α 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 lo 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 chondrecyte 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, typeⅡ collagen and Sox-9 expression further reduced, and type X collagen and matrix metalloproteinase 13 expression further increased. Compared with normal saline gr

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 

分 类 号：R318[医药卫生—生物医学工程]