基质金属蛋白酶与大骨节病的相关性研究  

Correlation of matrix metalloproteinases and Kashin-Beck disease

作　　者：陈静宏[1] 曹峻岭[1] 王治伦[1] 马天有[1] 王梦莹[1] 何颖[1] 杨占田[1] 陈晨[1] 

机构地区：[1]西安交通大学医学院地方病研究所及环境与疾病相关性基因教育部重点实验室,710061

出　　处：《中华地方病学杂志》2014年第4期357-362,共6页Chinese Journal of Endemiology

基　　金：国家自然科学基金（81273006、30872187）;教育部留学回国人员启动基金（11回国基金01）

摘　　要：目的：观察基质金属蛋白酶（Matrix metalloproteinases，MMPs）在大骨节病（Kashin-Beck disease， KBD）儿童软骨和低硒条件下T-2毒素中毒大鼠软骨中的表达，以及T-2毒素对软骨细胞MMP-13启动子的激活作用，探讨MMPs与KBD发病的关系。方法以5例尸检KBD儿童（KBD组）和5例因车祸死亡或畸形手术的正常儿童（对照组）手指节软骨为研究对象，采用免疫组织化学染色的方法，检测软骨中MMP-1和MMP-13的表达。雄性健康SD大鼠32只，按体质量采用随机数字表法分为常规饲料组、低硒饲料组，每组各16只。常规饲料硒含量为101.500μg/kg，低硒饲料硒含量为1.118μg/kg。大鼠低硒动物模型建造成功后，常规饲料组分为常规组和常规＋T-2组，低硒饲料组分为低硒组和低硒＋T-2组，每组8只。 T-2毒素（100μg·kg-1·d-1）灌胃饲养，30 d后处死大鼠，取左侧膝关节，用免疫组织化学染色方法检测大鼠关节软骨细胞MMP-1和MMP-13的表达水平。体外培养人软骨细胞系C28/I2细胞，分成空载体组、MMP-13启动子载体组、MMP-13启动子载体＋T-2毒素20μg/L组、MMP-13启动子载体＋T-2毒素40μg/L组，将-1602/＋20-MMP13-Luc启动子重组荧光素酶报导质粒以及空载体瞬时转染C28/I2细胞，24 h后，加入20μg/L和40μg/L T-2毒素，继续培养24 h，用荧光素酶报告基因检测T-2毒素对MMP-13启动子的作用。结果 KBD组儿童关节软骨表层和中层MMP-1表达阳性率[（29.73±10.12）%、（28.27±0.91）%]均高于对照组[（2.47±0.11）%、（0.00±0.00）%，P均〈0.05]，KBD组儿童关节软骨表层和中层 MMP-13表达阳性率[（13.21±4.32）%、（41.85±6.32）%]明显高于对照组[（5.72±0.31）%、（0.00±0.00）%，P均〈0.05]。低硒＋T-2毒素组大鼠关节软骨MMP-13在表层和中层表达水平[（13.21±4.32）%、61.85±8.68）%]明显高于常规组和低硒组[（2.43±0.22）%、（5.89±0.69）%，（3.03±0.29）Objective To investigate the expressions of matrix metalloproteinases（MMPs） in Kashin-Beck disease（KBD） cartilage as well as in a KBD rat model of T-2 toxin poisoning under selenium deficient conditions, and to investigate the effect of T-2 toxin on MMP-13 expression in human chondrocytes in vitro in order to determine a possible mechanism underlying KBD. Methods Samples of articular cartilage were divided into 2 groups：controls（samples from 5 normal children, traffic accident or operation）, and KBD（samples from 5 children with KBD, auctopsy）. Thirty-two Sprague-Dawley rats were divided into two groups by body weight using random number table： normal diet group（n = 16） and selenium-deficient diet group（n=16）. The selenium level in normal diet was 101.500μg/kg, and in selenium-deficient diet was 1.118μg/kg. Rats were fed for 4 weeks with selenium-deficient or normal diet, respectively. After successful build up of the low selenium rat model, normal diet group was then subdivided into 2 sub-groups： normal group（n = 8） and normal diet plus low T-2 toxin group（n = 8）;and selenium-deficient diet group was also subdivided into 2 sub-groups： selenium-deficient group （ n = 8 ） and selenium-deficient diet plus T-2 toxin group （ n = 8 ） . T-2 toxin of 100 μg·kg-1·d-1 was administered by intragastric administration for 30 days. Then the rats were sacrificed, and their knee joints were processed for histopathological evaluation. MMP-1 and MMP-13 locations in cartilages were performed by inmmunohistochemistry. Human chondrocytes C28/I2 were cultured in vitro. The experiment was divided into 4 groups： empty vector plasmid group, MMP-13 promoter plasmid group, MMP-13 promoter plasmid plus 20 μg/L T-2 toxin group and MMP-13 promoter plasmid plus 40 μg/L T-2 toxin group. MMP-13-luciferase reporter plasmid and vector plasmid were transiently transfected into C28/I2 cells for 24 hours, and then treated with 20 - 40 μg/L T-2 toxin for 24 hours. Transactivation of human MMP-13

关 键 词：大骨节病 基质金属蛋白酶 T-2毒素 硒 

分 类 号：R684.1[医药卫生—骨科学]