ADAM12异常表达对大骨节病患者软骨细胞损伤及IGFBP相关基因的影响  

Effects of ADAM12 abnormal expression on chondrocyte injury and IGFBP-related genes in patients with Kashin-Beck disease

作　　者：成博伦 杨正军[2] 常虹 郭雄 张峰 许鹏[3] 贾雨萌 Cheng Bolun;Yang Zhengjun;Chang Hong;Guo Xiong;Zhang Feng;Xu Peng;Jia Yumeng(chool of Public Health,Xi'an Jiaotong University Health Science Center,Key Laboratory of Trace Elements and Endemic Diseases,National Health Commission of the People's Republic of China,Collaborative Innovation Center of Endemic Diseases and Health Promotion in Silk Road Region,Xi'an 710061,China;Research Laboratory of Kashin-Beck Disease and Keshan Disease,Shaanxi Institute for Endemic Disease Prevention and Control,Xi'an 710003,China;Department of Osteonecrosis and Arthroplasty,Joint Disease Hospital,Honghui Hospital Affiliated to Xi'an Jiaotong University,Xi'an 710054)

机构地区：[1]西安交通大学医学部公共卫生学院,国家卫健委微量元素与地方病研究重点实验室,丝路区域地方病与健康促进协同创新中心,西安710061 [2]陕西省地方病防治研究所大骨节病克山病防治研究室,西安710003 [3]西安交通大学附属红会医院关节病医院骨坏死与关节重建科,西安710054

出　　处：《中华地方病学杂志》2023年第5期369-375,共7页Chinese Journal of Endemiology

基　　金：国家重点研发计划(2022YFC2503100)国家自然科学基金(81673112、82103959)。

摘　　要：目的探讨解整合素金属蛋白酶12(ADAM12)基因在大骨节病患者软骨细胞损伤中的作用及其对胰岛素样生长因子结合蛋白(IGFBP)相关基因的影响。方法由就诊于西安交通大学附属红会医院的5例大骨节病患者和5例对照受试者获得关节软骨样本,体外提取并培养关节软骨细胞。分别采用实时荧光定量PCR(qRT-PCR)和蛋白质印迹法检测大骨节病患者与对照受试者软骨细胞ADAM12 mRNA和蛋白表达水平。然后,采用慢病毒进行大骨节病患者软骨细胞ADAM12基因过表达实验,MTT法检测ADAM12基因过表达后细胞活性变化,并采用qRT-PCR检测软骨细胞分化相关基因Y染色体性别决定区-盒转录因子9(SOX9)、Ⅱ型胶原(COLⅡ),凋亡相关基因B细胞淋巴瘤/白血病-2相关X蛋白(BAX)以及合成代谢相关基因IGFBP3、IGFBP5 mRNA表达水平。结果大骨节病患者软骨细胞ADAM12 mRNA和蛋白表达水平(0.57±0.05、0.81±0.07)均显著低于对照受试者(1.00±0.00、1.00±0.00),差异均有统计学意义(t=-24.50、-3.61,均P<0.05)。MTT法结果显示,ADAM12过表达组软骨细胞活性(1.09±0.05)高于空载体对照组(1.00±0.08),差异有统计学意义(t=4.12,P=0.031)。qRT-PCR结果显示,与空载体对照组比较,ADAM12过表达组IGFBP3(2.35±0.79比0.96±0.25)、IGFBP5(2.13±0.30比0.98±0.34)、SOX9(2.92±0.51比0.94±0.36)和COLⅡmRNA表达水平(6.45±2.81比0.87±0.19)均较高,差异均有统计学意义(t=3.19、5.16、6.27、4.10,均P<0.05);而BAX mRNA表达水平(0.31±0.06比1.02±0.22)较低,差异有统计学意义(t=-11.16,P<0.001)。结论ADAM12基因在大骨节病患者软骨细胞损伤中可能有抑制凋亡和促进分化的作用,其过表达能够使IGFBP3和IGFBP5表达升高。Objective To investigate the role of a disintegrin and metalloprotease 12(ADAM12)gene in chondrocyte injury in patients with Kashin-Beck disease(KBD)and its impact on genes related to insulin-like growth factor binding protein(IGFBP).Methods Articular cartilage samples were obtained from 5 patients with KBD and 5 control subjects admitted to Honghui Hospital Affiliated to Xi'an Jiaotong University.Chondrocytes were extracted and cultured in vitro.Quantitative real-time PCR(qRT-PCR)and Western blotting were used to detect the expression levels of ADAM12 mRNA and protein in chondrocytes of patients with KBD and control subjects,respectively.Subsequently,ADAM12 gene overexpression was performed using lentivirus in chondrocytes of patients with KBD.MTT assay was used to detect changes in cell viability after ADAM12 gene overexpression,and qRT-PCR was used to detect the mRNA expression levels of chondrocyte differentiation related genes SRY-box transcription factor 9(SOX9)and type Ⅱ collagen(COLⅡ),apoptosis-related gene B-cell lymphoma/leukaemia-2-associated X protein(BAX),and anabolic related genes IGFBP3 and IGFBP5.Results The expression levels of ADAM12 mRNA and protein in chondrocytes of patients with KBD(0.57±0.05,0.81±0.07)were significantly lower than those of control subjects(1.00±0.00,1.00±0.00),and the differences were statistically significant(t=-24.50,-3.61,P<0.05).The results of MTT assay showed that the cell viability of chondrocytes in ADAM12 overexpression group(1.09±0.05)was higher than that in empty vector control group(1.00±0.08),and the difference was statistically significant(t=4.12,P=0.031).The results of qRT-PCR showed that compared with empty vector control group,the mRNA expression levels of IGFBP3(2.35±0.79 vs 0.96±0.25),IGFBP5(2.13±0.30 vs 0.98±0.34),SOX9(2.92±0.51 vs 0.94±0.36)and COLⅡ(6.45±2.81 vs 0.87±0.19)in ADAM12 overexpression group were significantly increased,and the differences were statistically significant(t=3.19,5.16,6.27,4.10,P<0.05);while the expression lev

关 键 词：大骨节病 解整合素金属蛋白酶12 软骨细胞 胰岛素样生长因子结合蛋白 

分 类 号：R684[医药卫生—骨科学]