低氧通过HIF-1α调控髓核细胞中ADAMTS-4和ADAMTS-5的表达  

Regulation of HIF-1α dependent hypoxia on expression of ADAMTS-4 and ADAMTS-5 in nucleus pulposus cells

作　　者：华东方[1] 徐敏铭 杨晨[1] 田野[1] 高阳[1] 魏磊鑫[1] 曹鹏[1] 袁文[1] 

机构地区：[1]第二军医大学附属长征医院脊柱外科,上海200003 [2]江西省上饶平安医院脊柱外科,江西334000

出　　处：《脊柱外科杂志》2017年第4期233-238,共6页Journal of Spinal Surgery

基　　金：国家自然科学基金青年项目(81501918);上海市卫计委面上项目(201440513)

摘　　要：目的观察低氧环境及低氧诱导因子1α(HIF-1α)对髓核细胞中解聚蛋白样金属蛋白酶(ADAMTS)-4和ADAMTS-5启动子活性的影响,探讨可能影响椎间盘退变的分子机制。方法将体外培养的大鼠髓核细胞分别置于常氧和低氧(1%O2)培养箱培养,采用蛋白质印迹分析及PCR方法检测细胞中ADAMTS-4和ADAMTS-5的表达。对获取的ADAMTS-4和ADAMTS-5启动子片段测序,并使用JASPAR数据库分析其中是否存在HIF-1α的结合位点。将HIF-1α过表达质粒、PGL3-ADAMTS-4质粒和PGL3-ADAMTS-5质粒转染至大鼠髓核细胞中,用双荧光素酶报告基因检测系统检测髓核细胞中HIF-1α对ADAMTS-4和ADAMTS-5基因启动子的调控作用。结果蛋白质印迹分析和PCR结果显示,低氧分别在蛋白质水平和m RNA水平抑制髓核细胞中ADAMTS-4、ADAMTS-5的表达。使用JASPAR软件分析后,发现ADAMTS-4启动子中可能包含1个HIF-1α的结合位点,而ADAMTS-5启动子中可能包含2个HIF-1α的结合位点。双荧光素酶报告基因检测结果显示,HIF-1α的过表达能够显著抑制ADAMTS-4和ADAMTS-5启动子活性。结论低氧可能通过HIF-1α抑制ADAMTS-4和ADAMTS-5的表达,保持椎间盘内环境的稳态,延缓椎间盘退变的发生。Objective To observe the effects of hypoxia and hypoxia inducible factor 1α（HIF-1α） on the activity of a disintegrin and metalloproteinase with thrombospondin motifs（ADAMTS）-4 and -5 promoter in the nucleus pulposus cells explor the molecular mechanism of intervertebral disc degeneration. Methods Western blotting and PCR were used to detect the expression of ADAMTS-4 and -5 in the nucleus pulposus cells in normoxia and hypoxia environments in vitro. The ADAMTS-4 and -5 promoters were sequenced,and the binding sites for the presence of HIF-1α were analyzed using JASPAR database. HIF-1α,PGL3-ADAMTS-4 and -5 plasmids were transfected into rat nucleus pulposus cells,and the effect of regulation of HIF-1α on ADAMTS-4 and -5 promoter were analyzed by Dual-Luciferasetm Reporter Assay System. Results The results of Western blotting and PCR showed that the expression of ADAMTS-4 and -5 in nucleus pulposus cells was suppressed by hypoxia at protein level and mRNA level,respectively. Using JASPAR software analysis,we found that the ADAMTS-4 promoter may contain 1 HIF-1α binding site,and the ADAMTS-5 promoter may contain 2 HIF-1α binding sites. Dual-Luciferasetm Reporter Assay System showed that overexpression of HIF-1α could significantly inhibit the transcriptions of ADAMTS-4 and -5. Conclusion Hypoxia may suppress the expression of ADAMTS-4 and -5 by HIF-1α,which may help to maintain the homeostasis of the intervertebral disc and delay the occurrence of disc degeneration.

关 键 词：芳香烃受体核转位子 金属蛋白酶类 细胞低氧 脊髓 

分 类 号：R651.21[医药卫生—外科学]