独活寄生汤调控对大鼠椎间盘软骨细胞Wntβ―catenin信号通路影响.docVIP

独活寄生汤调控对大鼠椎间盘软骨细胞Wntβ―catenin信号通路影响 　　【摘 要】目的：??Wnt/β-catenin信号通路探讨独活寄生汤水提物对大鼠椎间盘退变软骨细胞功能的影响。方法：①用水提加热回流法制备独活寄生汤水提物成分；②选取4周龄健康雄性SD大鼠30只，采用机械-酶消化法分离大鼠椎间盘软骨组织，建立软骨细胞体外培养体系并进行鉴定；③RT-PCR、Western blot法分别检测经DKK-1抑制剂干预及（或）经白细胞介素-1β诱导的椎间盘软骨细胞Wnt4、GSK-3β、β-catenin mRNA与蛋白含量的表达。结果：①椎间盘软骨细胞经Ⅱ型胶原法染色后，阳性对照组胞浆区域浸染为棕黄色；②RT-PCR、Western blot检测结果示，与正常组比较，模型组Wnt4、GSK-3β、 　　β-catenin mRNA与蛋白含量表达显著升高（P 0.01）；与模型组比较，独活寄生汤水提物组（100，200，400 μg?mL-1）的Wnt4、GSK-3β、β-catenin mRNA与蛋白含量表达显著降低（P 0.01或P 0.05），其中以200 μg?mL-1组的表达量最低（P 0.01）。结论：独活寄生汤水提物组可通过调控Wnt/β-catenin信号通路，下调大鼠椎间盘退变关节软骨中Wnt4、GSK-3β、β-catenin mRNA与蛋白含量表达，从而延缓椎间盘软骨细胞退变。 　　【关键词】 椎间盘退变性疾病；椎间盘软骨细胞；独活寄生汤；Wnt/β-catenin信号通路；大鼠 　　Effect of Duhuo Jisheng Tang（独活寄生汤）on the Regulation of Rat Intervertebral Disc Chondrocyte Wnt/β-catenin Signaling Pathway 　　LIU Bo-ling，CHEN Qi-yong，FU Chang-long，LIU Shao-qiang，YE Xiao-wei，LIANG Gui-qing，YE Jin-xia，LI Xi-hai 　　【ABSTRACT】Objective：To investigate the effect of Duhuo Jisheng Tang（独活寄生汤）water extract on the regulation of rat intervertebral disc chondrocyte Wnt/β-catenin signaling pathway.Methods：①Duhuo Jisheng Tang water extract was made by the water extraction and reflux method.②A total of thirty four-week-old healthy male SD rats used to isolate intervertebral disc cartilage tissue by mechanical enzymatic digestion method to establish in vitro culture system of chondrocytes for identification.③RT-PCR，Western blot were used to detect the expression of contents of Wnt4，GSK-3β and β-catenin mRNA intervened by DKK-1 　　inhibitor and induced by interleukin -1β.Results：①After typeⅡcollagen staining，the cytoplasmic domain of the intervertebral disc cartilage cells in the positive control group was disseminated yellowish brown.②RT-PCR and Western blot detect results showed that compared with the normal group，the expression of contents of Wnt4，GSK-3β， 　　β-catenin mRNA and protein increased significantly in the model group（P 0.01）.Compared with the model group，the expression of contents of Wnt4