骨性关节炎水通道蛋白1的表达与软骨细胞凋亡相关性研究

CORRELATION BETWEEN EXPRESSION OF AQUAPORINS 1 AND CHONDROCYTE APOPTOSIS IN ARTICULAR CHONDROCYTE OF OSTEOARTHRITIS

目的水通道蛋白1(aquaporins 1,AQP-1)表达变化可能与细胞凋亡相关。通过观察软骨组织中AQP-1和半胱天冬氨酸蛋白酶3(Caspase-3)的表达,探讨AQP-1表达与软骨细胞凋亡的相关性,为进一步研究骨性关节炎(os teoarthritis,OA)的发病机制提供实验依据。方法取8周龄清洁级雄性SD大鼠72只,体重286～320 g,平均300 g;随机分为手术组、假手术组和对照组,每组24只。手术组采用切断前交叉韧带及内侧副韧带、部分切除内侧半月板方法制备大鼠双膝OA模型;假手术组仅暴露关节腔;对照组不作处理。造模后观察大鼠一般情况,于1、2、4、8周处死大鼠取膝关节标本行大体、组织学观察;采用实时荧光定量PCR检测AQP-1和Caspase-3 mRNA的表达情况;使用酶标仪检测Caspase-3蛋白酶活性;并对AQP-1 mRNA表达与Caspase-3 mRNA的表达及蛋白酶活性的相关性进行分析。结果术后共6只大鼠死亡,均给予补充;其余大鼠均存活至实验完成。各时间点对照组及假手术组膝关节软骨外观及结构均正常;随时间延长手术组关节软骨表面粗糙且有裂隙,可见大量赘生物,软骨表层纤维化,细胞排列紊乱。参照Mankin评分标准,造模后1周各组组织学评分比较,差异均无统计学意义(P>0.05);2、4、8周时手术组与对照组、假手术组间比较,差异有统计学意义(P<0.05)。造模后1周,手术组AQP-1、Caspase-3 mRNA表达和Caspase-3蛋白酶活性与假手术组及对照组比较,差异均无统计学意义(P>0.05);而2、4、8周时手术组以上指标均明显高于对照组和假手术组(P<0.05),且随时间延长呈增高趋势。AQP-1 mRNA和Caspase-3 mRNA表达成正相关(r=0.817,P=0.000),回归方程为y=0.426 7x2+0.051 5x;AQP-1 mRNA表达和Caspase-3蛋白酶活性亦成正相关(r=0.945,P=0.000),回归方程为y=15.423 0x+4.392 8。结论 AQP-1的表达上调可能与软骨细胞凋亡相关,在OA发病过程中AQP-1表达的变化可能参与了OA的发生、发展。

Objective The changes of the aquaporins 1 （AQP-1） expression may be related to the chondrocyte apoptosis. To explore the correlation between the expression of AQP-1 and chondrocyte apoptosis by observing the expression of the AQP-1 and the Caspase-3, so as to provide experimental evidence for the further study in the pathogenesis of osteoarthritis （OA）. Methods Seventy-two 8-week-old clean grade male Sprague Dawley rats, weighing 286-320 g （mean, 300 g）, were randomly divided into the operated group （n=24）, the sham-operated group （n=24）, and the control group （n=24）. OA models were made by amputating the anterior cruciate l igament and medial collateral l igament, and partial excision of medial meniscus in operated group; the articular cavity was exposed only in sham-operated group; and no treatment was given in control group. The general condition of the rat was observed after model was establ ished. At 1, 2, 4, and 8 weeks, the specimens of knee joints were harvested to perform the gross and histological observations; the mRNA expressions of AQP-1 and Caspase-3 were determined by real-time fluorescent quantitative PCR; and the activity of the Caspase-3 protease was detected. The correlations between the expression of AQP-1 mRNA and the expressions of Caspase-3 mRNA and protease were analyzed. Results Totally 6 rats died after operation, and the rats were suppl ied immediately; the other rats survived to the end of experiment. The appearance and structure of knee articular cartilage were normal in control group and sham-operated group. While in operated group, the cartilage had a rough surface with fissure and vegetation, and fibrosis and irregular cell arrangement were seen on the surface of cartilage. There were significant differences in the Mankin score between the operated group and sham-operated group, control group at 2, 4, and 8 weeks （P 〈 0.05）. There was no significant difference in expressions of the AQP-1 mRNA and Caspase-3 mRNA, and the activity of the Caspase-3 protease among 3 groups at 1 week after operation （P 〉 0.05）; while the expressions of the AQP-1 mRNA, Caspase-3 mRNA, and the activity of the Caspase-3 protease in operated group were significantly higher than those in sham-operated group and control group at 2, 4, and 8 weeks after operation （P 〈 0.05）, and there was an increased trend over time. There was significantly positive correlation （r=0.817, P=0.000） between the expressions of AQP-1 mRNA and Caspase-3 mRNA, and the regression equation was y=0.426 7x2＋0.051 5x; meanwhile, there was also significantly positive correlation （r=0.945, P=0.000） between the expression of AQP-1 mRNA and the activity of Caspase-3 protease, and the regression equation was y=15.423 0x＋4.392 8. Conclusion The up-regulation of AQP-1 expression in OA cartilage may be related to the chondrocyte apoptosis, and the changes of AQP-1 expression may involve in the pathogenesis of OA.