作　　者： (郑文伟）; (何晓娟）; (贾良良）; (马玉环）; (陈后煌）; (邵翔）; (陈达）; (刘献祥）; (李西海）; (叶蕻芝）;

机构地区： 福建中医药大学,福建福州350122

出　　处： 《中医正骨》 2017年第8期8-16,共9页

摘　　要： 目的:探讨跳骨片含药血清抑制脂多糖诱导的软骨细胞炎症反应的作用机制。方法:将10只8周龄雄性SD大鼠随机分为跳骨片组和空白组,跳骨片组以0.32 g·kg-1剂量的跳骨片灌胃,空白组给予等量生理盐水灌胃;每天灌胃1次,连续7 d;末次灌胃1 h后,经腹主动脉取血,分别制备跳骨片含药血清和空白血清,低温保存备用。从10只4周龄SD大鼠膝关节软骨中分离软骨细胞并培养,光学显微镜下观察软骨细胞形态,并用Ⅱ型胶原酶免疫组化鉴定。将培养好的第2代软骨细胞随机分为空白血清组、模型组、跳骨片含药血清组,其中空白血清组以含10%空白血清的培养基(dulbecco modified eagle medium,DMEM)培养;模型组以浓度为10 ng·mL-1的脂多糖和含10%空白血清的DMEM培养;跳骨片含药血清组以浓度为10 ng·mL-1的脂多糖和含10%跳骨片含药血清的DMEM培养;3组均连续干预培养8 h,采用酶联免疫吸附法检测软骨细胞基质金属蛋白酶(matrix metalloproteinase,MMP)3、MMP9含量,采用荧光定量RT-PCR法检测软骨细胞中Wnt/β-catenin信号通路相关基因表达水平,采用Western blot法检测软骨细胞中β-链蛋白(β-catenin)、卷曲蛋白-2(Frizzled-2)的蛋白表达量,采用免疫荧光检测法检测软骨细胞中β-catenin、糖原合成酶激酶-3β(glycogen synthasc kinase-3β,GSK-3β)、蛋白多糖1(proteoglycans 1,PGS1)的蛋白表达量。结果:(1)软骨细胞免疫组化鉴定结果。第2代软骨细胞胞浆及细胞膜呈棕黄色阳性染色,具有典型的软骨细胞生物学特征。(2)软骨细胞MMP3、MMP9含量。脂多糖干预8 h后,空白血清组、模型组和跳骨片含药血清组的软骨细胞MMP3、MMP9含量比较,组间差异均有统计学意义[(34.019±1.036)ng·mL-1,(44.645±2.473)ng·mL-1,(32.941±1.792)ng·mL-1,F=36.060,P=0.000;(1.348±0.038)ng·mL-1,(1.562±0.112)ng·mL-1,(1.331±0.015)ng·mL-1,F=11.319,P=0.000];模型组软骨细胞MMP3、MMP9含量均高于空白血� Objective：To explore the mechanism of action of Tiaogu Pian（ 跳骨片, TGP）medicated serum in inhibiting inflammatory reaction induced by lipopolysaccharides in chondrocytes. Methods： Ten 8 - week - old male SD rats were randomly divided into TGP group and blank group. The rats in TGP group were intragastric administrated with TGP in dosage of 0.32 g/kg, while the others in blank group were intragastric administrated with the same dose of normal saline, once per day for 7 consecutive days. At 1 hour after the last intragastric administration, their blood were fetched out from abdominal aorta and were made into TGP medicated serum and blank serum respectively and the serum were reserved at low temperature for future use. The chondrocytes of ten 4 - week - old SD rats were isolated from knee artic- ular cartilage and were cultured. The chondrocytes morphology were observed under optical microscope, and immunohistochemical identifica- tion were carried out by using type 11 collagenase. The second - generation chondrocytes were randomly divided into blank serum group, mod- el group and TGP medicated serum group. The chondrocytes in blank serum group were cultured in dulbecco modified eagle medium （DMEM） supplemented with 10% blank serum. The chondrocytes in model group were cultured in DMEM supplemented with lipopolysac- charide（ LPS）with concentration of 10 ng/ml and 10% blank serum. The chondrocytes in TGP medicated serum group were cultured in DMEM supplemented with LPS with concentration of 10 ng/ml and 10% TGP medicated serum. The chondrocytes in the 3 groups were in- tervened and cultured for continuous 8 hours. The content of matrix metalloproteinase（MMP） 3 and MMP9 in chondrocytes were detected by using enzyme- linked immunoadsordent assay（ ELISA ）. The expression levels of gene related to Wnt/[3- catenin signaling pathway in chondrocytes were detected by using fluorescence quantitative RT - PCR method. The protein expressions of β - catenin and Frizzled - 2 in chondrocytes were dete

关 键 词： 骨关节炎 软骨细胞 跳骨片 软骨退变 信号通路 脂多糖类 基质金属蛋白酶类 炎症反应 动物实验