低氧诱导因子1α（HIF-1α）的SUMO化修饰调控对髓核细胞氧张力耐受的影响及机制研究

项目名称： 低氧诱导因子1α（HIF-1α）的SUMO化修饰调控对髓核细胞氧张力耐受的影响及机制研究

项目编号： No.81201423

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 医学四处

项目作者： 王锋

作者单位： 东南大学

项目金额： 23万元

中文摘要： 髓核细胞具有组成性表达低氧诱导因子-1α（HIF-1α）并耐受氧张力波动的特性。蛋白水平的类泛素（SUMO）化修饰机制与HIF-1α的转录后稳定及生物学效应密切相关。本研究观察正常氧与低氧状态下髓核细胞内SUMO化E1激活酶、E2连接酶和SUMO特异性蛋白酶的表达差异，探讨氧张力波动环境中髓核细胞SUMO化修饰活性的变化规律。继而强化或抑制髓核细胞SUMO化信号，观察HIF-1α的SUMO化水平与HIF-1α蛋白稳定性之间的量效关系，并结合HIF-1α下游信号Glut-1、VEGF的激活水平，综合评价SUMO化修饰调控对髓核细胞应答氧张力波动能力的影响。从"细胞调控SUMO化活性→维持HIF-1α蛋白稳定→适应氧张力波动"角度阐明髓核细胞对局部氧浓度变化的应答与耐受机制，为理解椎间盘退行性变的细胞学病因，以及通过干预蛋白SUMO化修饰信号来防治椎间盘退变打开崭新思路。

中文关键词： 椎间盘；类泛素化修饰；低氧；低氧诱导因子；翻译后修饰

英文摘要： Hypoxia inducible factor-1α (HIF-1α) is constitutively expressed in nucleus pulposus (NP) cells and shows a characteristic of normoxic stabilization, which indicates a metabolic adaptation to the intervertebral disc (IVD) microenvironment. Post-translational modification by members of small ubiquition-related modifier (SUMO) family regulates the stability and transactivation of HIF-1α， but the regulatory mechanism of the SUMO pathway has not been fully studied in NP cells. In this study rat NP cells will be cultured under hypoxic (2%O2) or normoxic (21%O2) conditions, and expressions of SUMO E1 activating enzymes, E2 conjugating enzymes, as well as sentrin-specific proteases (SENPs) will be evaluated. We assume that NP cells probably shift the balance between sumoylation and de-sumoylation in response to varied oxygen tensions. For modulating the SUMO pathway in NP cells, SENP1 specific siRNA and pCDNA/SENP1 plasmid are created. Gain- and loss-of-function experiments will be performed to investigate the relationship between HIF-1α sumoylation, HIF-1α protein stability, and the transcriptional activity of HIF-1α target genes e.g. VEGF, Glut-1. It is probable that NP cells stabilize the expression of HIF-1α by modulating the SUMO pathways, maintain a reasonable transcriptional activity of its downstream targets, t

英文关键词： intervertebral disc；sumoylation；hypoxia；hypoxia inducible factor；post translational modulation