不同氧浓度下髓核细胞中PI3K/Akt/HIF-1α信号通路表达对延缓椎间盘退变的意义  

Significance of PI3K/Akt/HIF-1αsignaling pathway expression in nucleus pulposus cells at different oxygen concentrations in delaying intervertebral disc degeneration

作　　者：周明瀚 张慧[1] 郑先波[1] 徐无忌[1] Zhou Minghan;Zhang Hui;Zheng Xianbo;Xu Wuji(Department of Orthopedics,the Second Affiliated Hospital of Hunan University of Chinese Medicine,Changsha 410208,Hunan Province,China)

机构地区：[1]湖南中医药大学第二附属医院骨伤二科,湖南省长沙市410208

出　　处：《中国组织工程研究》2024年第28期4491-4497,共7页Chinese Journal of Tissue Engineering Research

基　　金：湖南省自然科学基金项目(2022JJ30449),项目负责人:徐无忌;湖南省教育厅科学研究重点项目(21A0249),项目负责人:徐无忌;湖南中医药大学中医学一流学科开放基金项目(2022ZYX25),项目负责人:徐无忌。

摘　　要：背景:椎间盘退变是一种由于细胞凋亡、氧化应激、炎症反应等多种因素共同作用导致的疾病,目前认为其核心在于髓核细胞退变与凋亡,然而具体病理机制尚不明确。目的:通过探究不同氧浓度下髓核细胞中磷脂酰激醇-3-激酶(phosphatidylinositol-3kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)/缺氧诱导因子1α(hypoxia-inducible factor 1-alpha,HIF-1α)信号通路的表达及髓核细胞凋亡情况,明确不同氧浓度下髓核细胞的生物学特性,探讨椎间盘退变的机制。方法:取人正常及退变髓核细胞,取第2代细胞用于影像学鉴定,取第3代细胞分别采用37℃、空气、100%湿度环境,100μmol/L CoCl_(2)浓度的Leibovitz’s培养基、100μmol/L H_(2)O_(2)浓度的10%胎牛血清培养基构建常氧、低氧及氧化应激环境,将细胞分为正常髓核细胞+低氧环境、正常髓核细胞+常氧环境、正常髓核细胞+氧化应激环境、退变髓核细胞+低氧环境、退变髓核细胞+常氧环境、退变髓核细胞+氧化应激环境6组;采用CCK-8法检测细胞活力及增殖情况,流式细胞术检测细胞凋亡率,RT-PCR、Western-blot检测PI3K、AKT、HIF-1α的蛋白及mRNA表达情况。结果与结论:①在不同的氧浓度下,正常及退变髓核细胞的增殖率随氧浓度升高而下降,凋亡率随氧浓度升高而上升(P<0.05),以正常髓核细胞+低氧环境为对照组,细胞正常与否对凋亡率影响极为显著(P<0.001),氧浓度对髓核细胞增殖率与凋亡率影响极为显著(P<0.001),细胞是否退变与氧浓度二者交互作用对髓核细胞增殖率与凋亡率影响显著(P<0.05)。②在不同的氧浓度下,正常髓核细胞中低氧环境下PI3K、AKT、HIF-1α的蛋白及mRNA表达最高,氧化应激环境下其次,常氧环境下最低;退变髓核细胞中3项指标的蛋白和mRNA表达随氧浓度升高而下降;正常髓核细胞中PI3K、Akt的蛋白、mRNA表达均显著高于退变髓核细胞(P<0.05)。以正常髓核BACKGROUND:Intervertebral disc degeneration is a condition caused by the combined effects of various factors,such as cellular apoptosis,oxidative stress,and inflammatory responses.Currently,it is believed that the core of this condition lies in the degeneration and apoptosis of nucleus pulposus cells(NPCs).However,the specific pathological mechanisms remain unclear.OBJECTIVE:By investigating the expression of the phosphatidylinositol-3 kinase(PI3K)/protein kinase B(Akt)/hypoxia-inducible factor 1-alpha(HIF-1α)signaling pathway and the apoptosis of NPCs under different oxygen concentrations,to clarify the biological characteristics of NPCs under varying oxygen levels,and to explore the mechanisms of intervertebral disc degeneration.METHODS:Normal and degenerated NPCs were collected.The second-generation cells were used for imaging identification.The third-generation cells were cultured in the following conditions:37°C,air,100%humidity.Leibovitz’s medium containing 100μmol/L CoCl_(2)and 10%fetal bovine serum medium containing 100μmol/L H_(2)O_(2)were used to create distinct oxygen concentration environments for the third-generation cells,allowing for the investigation of cellular responses and behaviors under normal,low oxygen,and oxidative stress conditions.The third-generation cells were divided into six groups:normal NPCs+hypoxia,normal NPCs+normoxia,normal NPCs+oxidative stress,degenerated NPCs+hypoxia,degenerated NPCs+normoxia,and degenerated NPCs+oxidative stress groups.Cell viability and proliferation were assessed using the cell counting kit-8 method.Cell apoptosis was detected using flow cytometry.RT-PCR and western blot assay were utilized to examine the protein and mRNA expression levels of PI3K,AKT,and HIF-1α.RESULTS AND CONCLUSION:At different oxygen concentrations,the cell proliferation rate of both normal and degenerated NPCs decreased as the oxygen concentration increased.Conversely,the apoptosis rate increased as the oxygen concentration rose(P<0.05).With the normal NPCs+hypoxia group as the

关 键 词：氧浓度 髓核细胞 细胞凋亡 氧化应激 生物学特性 信号通路 

分 类 号：R496[医药卫生—康复医学] R318[医药卫生—临床医学] R681