“风寒湿致痹”理论指导下体内外骨关节炎模型建立与评价研究

Establishment and Evaluation of Osteoarthritis Model in Vivo and in Vitro Under the Guidance of The Theory of"Wind-Cold-Dampness Causing Arthralgia"

目的:建立风寒湿型骨关节炎大鼠体内外模型,为“风寒湿致痹”这一中医病因病机理论提供实验基础。方法:动物模型的建立与评价方面,将SD大鼠随机分为空白组(Blank组)、膝骨关节炎组(KOA组)、风寒湿+膝骨关节炎组(FKOA组),采用改良Hulth法和人工气候箱模拟风寒湿环境构建风寒湿型膝骨关节炎动物模型,将FKOA组血清同时备用于体外研究,通过HE染色观察大鼠膝关节病理形态,ELISA法检测大鼠血清中白细胞介素(interleukin,IL)-1β、IL-6的含量。体外模型的建立与评价方面,通过分离、提取并培养原代软骨细胞,分为膝骨关节炎对照组(Control组,以IL-1β进行诱导)和风寒湿组(FHS组,以IL-1β进行诱导及FKOA组大鼠模型的血清干预)。显微镜下观察软骨细胞形态,通过基因测序对比与骨关节炎发生发展相关的一些基因的表达情况,并着重检测Ⅱ型胶原蛋白α1(collagen type II alpha 1,Col2a1)、蛋白多糖(aggrecan,Acan)、SRY相关高迁移率族盒蛋白转录因子9(SRY-related HMG box transcription factor 9,Sox9)mRNA表达的水平。结果:体内实验发现,与Blank相比较,其余2组的大鼠膝关节软骨细胞明显具有肥大化和成簇化表现,且FKOA组大鼠血清中IL-1β、IL-6含量较KOA组显著升高(P<0.05);体外实验中,相较Control组软骨细胞,加入风寒湿血清的软骨细胞形态学明显发生改变,多为扁平长梭形或不规则形,基因测序结果显示,与Control组比较,FHS组Col2a1、Acan、Sox9 mRNA的表达明显下降(P<0.01)。结论:风寒湿因素处理后的膝骨关节炎大鼠加重了膝骨关节炎的病症特点,值得进一步研究与发掘。

Objective:To establish a rat model of wind-cold-dampness osteoarthritis in vivo and in vitro,so as to provide experimental basis for the theory of etiology and pathogenesis of wind-cold-dampness in TCM.Methods:Establishment and evaluation of animal model:SD rats were randomly divided into blank group(Blank group),knee osteoarthritis group(KOA group)and wind-cold-damp knee osteoarthritis group(FKOA group).The animal model of wind-cold-damp knee osteoarthritis was established by modified Hulth method and artificial climate box simulating wind-cold-damp environment.The serum of FKOA group was prepared for in vitro study at the same time.The pathological morphology of knee joint was observed by HE staining,and the contents of IL-1βand IL-6 in serum were detected by ELISA.Establishment and evaluation of in vitro model:primary chondrocytes were isolated,extracted and cultured and divided into knee osteoarthritis control group(Control group,induced by IL-1β)and wind-cold-dampness group(FHS group,induced with IL-1βand serum intervention of FKOA group).The morphology of chondrocytes was observed under microscope,and the expressions of some genes related to the occurrence and development of osteoarthritis were compared by gene sequencing,the mRNA expression levels of typeⅡcollagenα1(Col2a1),Aggrecan(Acan)and SRY-related HMG box transcription factor 9(Sox9)were detected.Results:compared with Blank group,the chondrocytes of knee joint in the other two groups showed obvious hypertrophy and clustering,and the contents of IL-1βand IL-6 in serum of rats in FKOA group were significantly higher than those in KOA group(P<0.05).In vitro experiment,compared with Control group,the morphology of chondrocytes treated with wind-cold-dampness serum changed significantly,most of them were flat,long fusiform or irregular shape,and the results of gene sequencing showed that the expression of Col2a1,Acan and Sox9 mRNA in FHS group was significantly lower than those in Control group(P<0.01).Conclusion:The knee osteoarthritis rats treated with wind-cold-dampness factors aggravate the characteristics of knee osteoarthritis,which is worthy of further study and exploration.