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**★

摘要

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 col ected 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 L 4-6 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. Fol owing intragastrical administration for 2 months, lumbar vertebra at L 4-6 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 cel s, and chondrocyte reduction. In addition, type II col agen and Sox-9 expression in intervertebral disc cartilage decreased, while type X col agen and matrix metal oproteinase 13 expression increased. At 4.5 months old, the cartilage injury was worsened, type II col agen and Sox-9 expression further reduced, and type X col agen and matrix metal oproteinase 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 col agen and Sox-9 expression, but decreased type X col agen and matrix metal oproteinase 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.%　　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。