作　　者： (何晓娟）; (林平冬）; (马玉环）; (翁霞萍）; (郑文伟）; (李西海）; (林洁）; (付长龙）; (邵翔）; (叶蕻芝）; (刘献祥）;

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1-7,共7页

摘　　要： 目的:探讨独活寄生汤含药血清抑制白细胞介素1β(interleukin-1 beta,IL-1β)诱导的软骨细胞炎症反应的作用机制。方法:将10只2月龄雄性SD大鼠随机分为正常组和独活寄生汤组,独活寄生汤组以9.3 g·kg^(-1)剂量的独活寄生汤灌胃,正常组给予等量生理盐水灌胃;每日灌胃2次,连续1周;末次灌胃后,经腹主动脉取血,分别制备独活寄生汤含药血清及空白血清,低温保存备用。截取6只4周龄SD大鼠膝关节软骨,采用酶消化法分离并培养软骨细胞,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。取生长良好的第2代软骨细胞,采用MTT法检测含药血清培养24 h、36 h、48 h、72 h后的软骨细胞活性,采用酶联免疫吸附法测定不同浓度IL-1β干预后软骨细胞的基质金属蛋白酶(matrix metalloproteinase,MMP)-13含量。将培养好的第2代软骨细胞随机分为空白血清组、模型组、独活寄生汤含药血清组,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;模型组加入浓度为15 ng·m L^(-1)的IL-1β干预24 h后,采用含10%空白血清的DMEM培养;独活寄生汤含药血清组加入浓度为15 ng·m L^(-1)的IL-1β干预24 h后,采用含10%独活寄生汤含药血清的DMEM培养;3组均连续培养48 h,采用Western blot法检测软骨细胞中G蛋白偶联信号传导系统关键调控因子的蛋白表达情况。结果:(1)软骨细胞形态及免疫组化鉴定结果。第2代软骨细胞胞浆丰富,细胞周围可见具有折光性的细胞外基质,细胞长势呈"铺路石"状,胞浆呈棕黄色阳性染色。(2)独活寄生汤含药血清培养不同时间后的软骨细胞活性。含药血清培养24 h、36 h、48 h、72 h后的软骨细胞活性比较,差异有统计学意义(1.01±0.01,1.03±0.02,1.07±0.01,1.02±0.02,F=8.300,P=0.008)。含药血清培养24 h时的软骨细胞活性低于培养48 h时的软骨细胞活性(t=-4.648,P=0.002);与培养36 h、72 Objective ：To explore the mechanism of action of Duhuo Jisheng Tang（独活寄生汤, DHJST） medicated serum in inhibiting inflammatory reaction induced by interleukin - 1 beta（ IL - 1β） in chondrocytes. Methods：Ten 2 - month - old male SD rats were randomly divided into normal group and DHJST group. The rats in DHJST group were intragastric administrated with DHJST in dosage of 9.3 g/kg, while the others in normal group were intragastric administrated with the same dose of normal saline, twice per day for 1 consecutive week. After the last intragastric administration, their blood were fetched out from abdominal aorta and were made into DHJST medicated serum and blank serum respectively and the serum were reserved at low temperature for future use. The knee articular cartilage of six 4 - week - old SD rats were fetched out and the chondrocytes were isolated and cultured by using enzymatic digestion method. The chondrocytes morphology were observed under optical microscope, and immunohistochemical identification were carried out by using type II collagenase. The well - growned second - generation chondrocytes were fetched out, and their cytoactive were detected by using MTr method after cultured in medi- cated serum for 24,36,48 and 72 hours, and the matrix metatloproteinase（MMP） - β content of chondrocytes were detected by using en- zyme -linked immunoadsordent assay（ELISA） after intervention with different concentrations of IL - 1β. The second - generation chondro- cytes were randomly divided into blank serum group, model group and DHJST medicated serum group. The chondrocytes in blank serum group were cultured in dulbecco modified eagle medium（DMEM） supplemented with 10% blank serum, while the chondrocytes in model group and DHJST medicated serum group were intervened by IL - 1β with concentration of 15 ng/ml for 24 hours and then were cultured in I）MEM supplemented with 10% blank serum and 10% DHJST medicated serum respectively. The protein expression of key regulating factor of G

关 键 词： 骨关节炎 软骨细胞 独活寄生汤 软骨退变 蛋白偶联信号通路 白细胞介素 基质金属蛋白酶 炎症反应 动物实验