作　　者： (贾峻）; (荣兵）; (李建）; (孙鹏）; (陈少青）;

机构地区： 天津中医药大学第一附属医院,天津300193

出　　处： 《中国实验方剂学杂志》 2017年第17期159-165,共7页

摘　　要： 目的:观察独活寄生汤含药血清对膝骨性关节炎(KOA)大鼠关节软骨细胞代谢、骨形成蛋白-7(BMP-7)及沉默信息调节因子相关酶1(SIRT1)的影响,为独活寄生汤的临床应用补充理论依据。方法:采用Hulth法建立KOA模型大鼠,模型成功后采用酶原消化法培养KOA软骨细胞;另将40只大鼠分为独活寄生汤低、中、高剂量组(0.85,1.7,3.4 g·kg^(-1))及硫酸氨基葡萄糖组(硫酸氨基葡萄糖胶囊0.3 g·kg^(-1)),灌胃14 d后处死取血清;将培养细胞分为正常组、模型组、独活寄生汤含药血清低、中、高剂量组(对应剂量组大鼠血清培养)及硫酸氨基葡萄糖胶囊组(硫酸氨基葡萄糖组大鼠血清培养);采用结晶紫法检测各组软骨细胞增殖能力;采用荧光原位末端转移酶标记法(TUNEL)法检测各组软骨细胞凋亡情况;实时荧光定量聚合酶链式反应(Real-time PCR)检测各组软骨细胞中基质金属蛋白酶-13(MMP-13),MMP-3,Ⅱ型胶原蛋白(ColⅡ),聚集蛋白聚糖(Aggrecan),BMP-7及SIRT1 mRNA的表达;蛋白质印迹法(Western blot)检测各组软骨细胞中MMP-13,MMP-3,ColⅡ,Aggrecan,BMP-7及SIRT1的表达。结果:(1)与正常组软骨细胞比较,模型组细胞增殖能力明显降低,与模型组细胞比较,独活寄生汤含药血清低、中、高剂量组及硫酸氨基葡萄糖组软骨细胞增殖能力提高(P<0.01);(2)与正常组软骨细胞比较,模型组细胞凋亡数量明显增加,与模型组软骨细胞比较,独活寄生汤低、中、高剂量组及硫酸氨基葡萄糖组细胞凋亡数量明显降低(P<0.01);(3)与正常组软骨细胞比较,模型组细胞MMP-13,MMP-3表达升高,ColⅡ,Aggrecan,BMP-7及SIRT1下降(P<0.01);与模型组比较,低剂量组细胞中MMP-13,MMP-3 mRNA及蛋白表达无明显变化;硫酸氨基葡萄糖组及中、高剂量组细胞中MMP-13,MMP-3 mRNA及蛋白表达明显下降,且剂量越高上述蛋白及mRNA表达越低;硫酸氨基葡萄糖组及低、中、高剂量组细胞中ColⅡ,Aggreca Objective ： To observe the effect of serum containing Duhuo Jisheng Tang on cell metabolism,bone morphogenetic protein-7 （BMP-7） and the silent mating type information regulation 2 homolog 1 （SIRT1） in articular cartilage of rats with knee osteoarthritis （KOA）, and complement the theory basis for the clinical application of Duhuo Jisheng Tang. Method： Hulth method was adopted to establish the KOA model rats, and after successful modeling, enzyme digestion method was used for primary culture of KOA cartilage cells. Forty rats were divided into Duhuo Jisheng Tang low, middle and high dose groups （0.85, 1.7, 3.4 g ·kg^-1 ） and glucosamine sulfate group （0.3 g·kg^-1）. After 14 days of intragastric administration, serum was taken. Cultured cells can be divided into normal control group, model group, Duhuo Jisheng Tang drug-containing serum groups （high, middle and low dose groups） and positive control group. Then crystal violet method was used to test the cell proliferation of each group; fluorescent TUNEL method was used to measure the cell apoptosis of each group. The mRNA expression levels of matrix metalloproteinase-13 （MMP-13） , MMP-3, Col Ⅱ , Aggrecan, BMP-7 and SIRT1 in chondrocytes were detected by Real-time PCR, and the protein expression levels of MMP-13, MMP-3, Col Ⅱ, Aggrecan, BMP-7 and SIRTI were detected by Western blot in each group of cartilage cells. Result： （1） As compared with the normal control group, the proliferation was significantly decreased in model group. As compared with the model group, the proliferation of chondrocytes was significantly increased in Duhuo Jisheng Tang low, middle, and high dose groups and positive control group （P 〈 0.01 ）. （2） As compared with the normal control group, the number of apoptotic cells was significantly increased in model group. As compared with model group, the apoptotic rate of chondrocytes was significantly decreased in Duhuo Jisheng Tang low, middle, high dose groups and positive control group （P �

关 键 词： 独活寄生汤 膝骨性关节炎 细胞代谢 软骨细胞 骨形成蛋白