大骨节病患者踝关节软骨IGF-1、IGFBP2基因表达的特征及意义

Characteristics and significance of IGF-1 and IGFBP2 gene expressions in ankle cartilage of patients with Kashin-Beck disease

目的探讨大骨节病(KBD)患者踝关节软骨胰岛素样生长因子1(IGF-1)、胰岛素样生长因子结合蛋白2(IGFBP2)基因表达的特征及意义。方法采用病例-对照研究方法,选择2010年1月至2016年12月在陕西省人民医院骨科住院的KBD患者10例为KBD组,并以同期因外伤致踝关节骨折但无距骨损伤的患者10例为对照组,收集两组患者软骨组织。分别采用免疫组织化学、实时荧光定量PCR及蛋白免疫印迹法检测患者软骨组织中IGF-1、IGFBP2阳性细胞、mRNA和蛋白表达情况。根据KBD患者踝关节软骨IGF-1、IGFBP2基因表达情况,在陕西省人民医院选择1例创伤致截肢患者,取踝关节软骨制备软骨细胞进行体外细胞验证实验;将软骨细胞分为对照组(0 ng/ml T-2毒素)、T-2处理组(20 ng/ml T-2毒素)、T-2+IGFBP2沉默组(20 ng/ml T-2毒素+50 nmol/L IGFBP2 siRNA),采用MTT法和二甲基亚甲基蓝染色法检测3组软骨细胞活性及硫酸糖胺多糖(sGAG)分泌情况。结果对照组与KBD组患者软骨组织IGF-1[(47.26±8.97)、(68.15±7.42)个]、IGFBP2阳性细胞数[(27.56±5.40)、(71.85±7.62)个]比较,差异均有统计学意义(t=4.487、9.402,P均<0.01);与对照组比较,KBD组患者软骨组织IGF-1、IGFBP2 mRNA和蛋白表达水平均较高,差异均有统计学意义(t=3.340、20.700,4.684、8.699,P<0.05或<0.01)。细胞实验中,对照组、T-2处理组、T-2+IGFBP2沉默组软骨细胞活性和sGAG含量比较,差异均有统计学意义(F=226.70、80.66,P均<0.01);其中,T-2处理组、T-2+IGFBP2沉默组细胞活性、sGAG含量均低于对照组(P均<0.05),且T-2+IGFBP2沉默组均高于T-2处理组(P均<0.05)。结论KBD患者踝关节软骨中IGF-1、IGFBP2基因表达明显较高。沉默IGFBP2基因能够降低T-2毒素对软骨细胞活性及sGAG分泌的抑制作用。

Objective To investigate the characteristics and significance of insulin-like growth factor 1(IGF-1)and insulin-like growth factor binding protein 2(IGFBP2)expressions in ankle cartilage of patients with Kashin-Beck disease(KBD).Methods In this case-control study,10 KBD patients who were hospitalized in the Department of Orthopedics of Shaanxi Provincial People's Hospital from January 2010 to December 2016 were selected as KBD group,and 10 patients with ankle fracture caused by trauma but without talus injury during the same period were selected as control group,the cartilage tissues of the two groups were collected.IGF-1,IGFBP2 positive cells,the mRNA and protein expressions of IGF-1,IGFBP2 in the cartilage tissues were detected by immunohistochemistry,real-time fluorescent quantitative PCR and Western blotting,respectively.According to the expressions of IGF-1 and IGFBP2 in ankle cartilage of KBD patients,a patient with amputation caused by trauma was selected in Shaanxi Provincial People's Hospital,and ankle joint cartilage was taken to prepare chondrocytes for in vitro cell verification experiments.The chondrocyte were divided into control group(0 ng/ml T-2 toxin),T-2 treatment group(20 ng/ml T-2 toxin)and T-2+IGFBP2 silenced group(20 ng/ml T-2 toxin+50 nmol/L IGFBP2 siRNA),the MTT method and dimethyl methylene blue staining were used to detect the activity of chondrocyte and the secretion of sulfated glycosaminoglycan(sGAG).Results In the control group and the KBD group,the number of IGF-1[(47.26±8.97),(68.15±7.42)cells]and IGFBP2 positive cells[(27.56±5.40),(71.85±7.62)cells]in the cartilage tissues were significantly different(t=4.487,9.402,P<0.01).Compared with the control group,the IGF-1,IGFBP2 mRNA and protein expression levels in KBD group were significantly higher,the differences were significantly different(t=3.340,20.700,4.684,8.699,P<0.05 or<0.01).In cell experiment,the chondrocyte activitives and sGAG contents of the control group,T-2 treatment group,and T-2+IGFBP2 silenced group were significantly different(F=226.70,80.66,P<0.01);among them,the cell activitives and sGAG contents of the T-2 treatment group and T-2+IGFBP2 silenced group were lower than those of control group(P<0.05),and the T-2+IGFBP2 silenced group were higher than those of the T-2 treatment group(P<0.05).Conclusions The expressions of IGF-1 and IGFBP2 in the ankle cartilage of KBD patients are significantly higher.Silencing IGFBP2 gene can reduce the inhibitory effect of T-2 toxin on chondrocyte activity and the secretion of sGAG.