缺氧诱导锌指蛋白A20表达抑制髓核细胞凋亡和炎症  

Hypoxia-induced expression of zinc finger protein A20 inhibits apoptosis and attenuates inflammation in nucleus pulposus cells

作　　者：郭帅池 刘豪 韩正瀚 刘雯隽 刘渤[1] GUO Shuaichi;LIU Hao;HAN Zhenghan;LIU Wenjun;LIU Bo(Department of Orthopedics,the First Affiliated,Hospital of Chongqing Medical University,Chongqing,400016,China)

机构地区：[1]重庆医科大学附属第一医院骨科,重庆400016

出　　处：《陆军军医大学学报》2023年第16期1693-1701,共9页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(81572202)。

摘　　要：目的探讨在缺氧条件下锌指蛋白A20对椎间盘髓核细胞的作用及其可能机制。方法分别在常氧(21%O_(2))和缺氧(1%O_(2))细胞培养箱培养大鼠椎间盘,并使用二甲基乙二酰基甘氨酸(dimethyloxalylglycine,DMOG)处理后分为常氧组、常氧+DMOG组、缺氧组和缺氧+DMOG组。通过组织切片染色和Western blot实验检测各组髓核组织形态学改变以及HIF-1α和A20蛋白表达差异;利用A20干扰腺病毒,分别在缺氧和常氧条件下处理人髓核细胞后分为缺氧+空载病毒组、缺氧+A20干扰腺病毒组、常氧+空载病毒组、常氧+A20干扰腺病毒组。分别通过流式细胞学、JC-1检测、EdU检测和蛋白免疫印迹实验检测各组髓核细胞凋亡、线粒体膜电位、增殖、细胞外基质以及炎性细胞因子表达差异。结果相比于常氧组,其余3组形态学上退变程度较轻,HIF-1α和A20蛋白表达量增加(P<0.05);相较于缺氧+空载病毒组,缺氧+A20干扰腺病毒组髓核细胞TNF-α和P-p65表达量增加(P<0.05),细胞凋亡增加(P<0.05),细胞增殖下降(P<0.05),细胞外基质蛋白表达量减少(P<0.05)。而在常氧+空载病毒组与常氧+A20干扰腺病毒组中,上述指标差异无统计学意义。结论缺氧可通过诱导A20蛋白表达,抑制髓核细胞的凋亡、炎症,促进髓核细胞增殖和细胞外基质合成。Objective To explore the effect and mechanism of zinc finger protein A20 in the nucleus pulposus cells of intervertebral disc with hypoxic conditions.Methods Rat intervertebral discs were cultured in normoxic(21%0_(2))and hypoxic(1%O_(2))conditions treated with dimethyloxalylglyeine(DMOG)and divided into normoxia group,normoxia+DMOG group,hypoxia group and hypoxia+DMOG group.Histological staining and Western blotting were used to detect the hi stomorphological changes of the nucleus pulposus and the differences in expression of HIF-1ou and A20.A20 interferi ng adenovirus were used to treat human nucleus pulposus cells with hypoxie and normoxic condition respectively,and divided into hypoxia+empty adenovirus group,hypoxia+A20 interference adenovirus group,normoxia+empty adenovirus group and normoxia+A20 interference adenovirus group.Flow cytometry,JC-1 detection,EdU detection and W estern botting were used to detect the diferences in apoptosis,mitochondrial membrane potential,prol iferation,extracellular matrix and inflammatory cytokine expression of nuceus pulposus cells.Results Compared with the normoxia group,the morphological degeneration of the other 3 groups was milder,and the protein levels of HIF-1c and A20 were significantly increased(P<0.05);Compared with the hypoxia+empty adenovirus group,the hypoxia+A20 interference adenovirus group showed the increased protein levels of TNF-C and P-p65 in mucleus pulposus cells(P<0.05),increased apoptosis(P<0.05),decreased cell proliferation(P<0.05)and decreased protein levels of extracellular matrix proteins(P<0.05).In the normoxia+empty adenovirus group and the normoxia+A20 interference adenovirus group,there was no significant difference of the indicators mentioned above.Conclusion Hypoxia can inhibit the apoptosis and attenuate inflammation and promote the proliferation and the anabolism of extracellular matrix in nucleus pulposus cells by inducing the expression of A20 protein.

关 键 词：锌指蛋白A20 椎间盘退变 体外器官培养 缺氧 髓核细胞 

分 类 号：R341[医药卫生—基础医学] R363.22R681.53