摘要目的 定量分析骨关节炎大鼠血清中氨基酸水平的动态变化,为进一步研究骨关节炎的生物标志物和代谢机制提供线索.方法 将60只雄性SD大鼠按体质量采用随机数字表法分为模型组和对照组,按实验周期分术后4、8、12周3个时间进行,每个时间模型组与对照组各10只大鼠.模型组用改良的Huhh法对左后肢膝关节进行手术,5d后,驱赶模型组大鼠使之自由活动,每天30 min.实验期间,两组大鼠均饲喂普通固体饲料,饮用自来水.实验期满,取膝关节进行组织病理学观察;利用高效液相色谱-四级杆离子阱串联质谱仪检测大鼠血清中氨基酸;用受试者工作特征(ROC)曲线下面积(AUC)来判定差异氨基酸对疾病的诊断能力,AUC为＞0.7～0.9时有一定准确性,＞0.9时有较高准确性.结果 光镜下,对照组大鼠关节软骨表层光滑、连续,细胞整体形态结构良好,层次分明;模型组大鼠关节软骨层变薄,表层粗糙、缺损,软骨细胞变性、坏死、消失,病变随观察周期延长而加重.共检出40种血清氨基酸,与对照组比较,术后4周模型组有24种氨基酸含量降低,14种升高,其中牛磺酸、谷氨酰胺、丝氨酸、谷氨酸、天门冬氨酸、γ-氨基丁酸、羟基脯氨酸组间比较差异有统计学意义(P均＜ 0.05);与对照组比较,术后8周模型组有24种氨基酸含量升高,16种降低,其中组氨酸、丝氨酸、谷氨酸、同型精氨酸、赖氨酸、异亮氨酸、甘氨酸组间比较差异有统计学意义(P均＜ 0.05);与对照组比较,术后12周模型组有36种氨基酸含量降低,其中21种氨基酸含量组间比较差异有统计学意义(P均＜0.05),AUC均≥0.740,有14种氨基酸AUC≥0.800,最高的为犬尿氨酸(0.980).结论 成功地建立了大鼠膝骨关节炎模型.随时间增加,骨关节炎病变加重,大鼠体内氨基酸代谢出现紊乱.

abstractsObjective To quantitatively analyze the dynamic changes of amino acid levels in serum of osteoarthritis rats,and to provide clues for further studies on biomarkers and metabolic mechanisms of osteoarthritis.Methods Sixty healthy male Sprague-Dawley (SD) rats were randomly divided into model group and control group according to their body weight by random number table method,the experimental period was 4,8 and 12 weeks,respectively,ten rats in each group.The left knee hind joints of rats in model group were operated by the modified Hulth method.After 5 days,rats of model group were driven to move for 30 minutes a day.Rats of two groups were fed ordinary solid fodder and drank tap water.At the end of the experimental period,the knee joints were collected for pathological observation.The amino acid content in serum was determined by high performance liquid chromatography-quadrupole ion trap tandem mass spectrometer (HPLC/Q-TRAP-MS/MS).The area under the receiver operating characteristic (ROC) curve (AUC) was used to determine the ability of differential amino acids to diagnose disease,AUC ＞ 0.7-0.9 was certain accuracy,＞ 0.9 was high accuracy.Results Under the light microscope,in control group,the articular cartilage surface was smooth and continuous,and the chondrocytes had good overall morphological structure and distinct layers;in model group,the articular cartilage of rats thinned,the surface was roughness and defect,the chondrocytes degenerated,necrosis and disappeared,and the lesions were aggravated with the extension of observation period.Forty kinds of amino acids were detected.Compared with control group,at 4 weeks after surgery,the level of 24 kinds of amino acids in model group decreased and 14 kinds of amino acids were increased,among which the differences in taurine,glutamine,serine,glutamic acid,aspartic acid,γ-aminobutyric acid and hydroxyproline were statistically significant (P ＜ 0.05);at 8 weeks after surgery,24 kinds of amino acids in model group were increased and 16 were decreased,among which the differences in histidine,serine,glutamic acid,homoarginine,lysine,isoleucine and glycine were statistically significant (P ＜ 0.05);at 12 weeks after surgery,36 kinds of amino acids in model group were decreased,among which the differences in 21 kinds of amino acids were statistically significant (P ＜ 0.05),all of their AUC were not less than 0.740,and the AUC of 14 amino acids were not less than 0.800,and the highest was kynurenine (0.980).Conclusions The model of knee osteoarthritis in rats is established successfully.With the increased time,the progress of osteoarthritis lesions increases,and the amino acid metabolism in osteoarthritis rats is out of order.