生理性拉应力通过Nell-1/Ihh信号通路对ATDC5软骨细胞分化的调控作用  

Regulatory effect of physiological tensile stress on differentiation of ATDC5 chondrocytes through Nell-1/Ihh signaling pathway

作　　者：董紫薇 齐慧川 马俊 薛晴 聂瑾涵 于航[1,2] 胡敏 DONG Ziwei;QI Huichuan;MA Jun;XUE Qing;NIE Jinhan;YU Hang;HU Min(Department of Orthodontics,Stomatology Hospital,Jilin University,Changchun 130021,China;Jilin Provincial Key Laboratory of Tooth Development and Jaw Bone Remodeling and Regeneration,Changchun 130021,China)

机构地区：[1]吉林大学口腔医院正畸科,吉林长春130021 [2]吉林省牙发育及颌骨重塑与再生重点实验室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2024年第1期1-9,共9页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金青年科学基金项目(82100956);吉林省科技厅自然科学基金项目(YDZJ202301ZYTS432);吉林省财政厅医疗卫生人才项目(jcsz2020304-22)。

摘　　要：目的:探讨生理性拉应力对软骨细胞分化的调控作用,并阐明其相关信号通路机制。方法:体外培养软骨ATDC5细胞,应用四点弯曲细胞力学加载仪对其施加生理性拉应力,首先分为对照组和拉应力组(2 000μstrain/2 h组),另分为不同力值(1 000、2 000和3 000μstrain)加力时间为2 h和力值为2 000μstrain不同加力时间(1、2和4 h)组,同时设未加力的细胞为对照组,采用实时荧光定量PCR (RT-qPCR)法检测各组细胞中Ⅱ型胶原(Col-Ⅱ)、Ⅹ型胶原(Col-Ⅹ)、聚集蛋白聚糖(Aggrecan)、性别决定区Y框蛋白9 (SOX9)、血管内皮生长因子(VEGF)、增殖细胞核抗原(PCNA)、Nel样1型分子(Nell-1)、Runt相关转录因子2 (Runx2)、印度刺猬因子(Ihh)、补缀同源物1 (Ptch-1)、GLI家族锌指蛋白1 (Gli-1)和刺猬因子相互作用蛋白1 (Hhip-1) mRNA表达水平,采用Western blotting法检测各组细胞中Nell-1、Runx2和Ihh蛋白表达水平。ATDC5细胞分为对照组、环巴胺组、拉应力组和环巴胺+拉应力组,采用RT-qPCR法检测各组细胞中Nell-1、Ihh、Ptch-1、Gli-1和Hhip-1 mRNA表达水平,采用Western blotting法检测各组细胞中Nell-1和Ihh蛋白表达水平。结果:与对照组比较,2 000μstrain/2 h组细胞中Col-Ⅱ、 Col-Ⅹ、 Aggrecan、 SOX9、VEGF和PCNA mRNA表达水平均明显升高(P<0.01)。在对细胞施加2 000μstrain不同加力时间(1、2和4 h)或不同力值(1 000、2 000和3 000μstrain) 2 h的拉应力后,与对照组比较,随时间的延长或力值的增加其他各组细胞中Runx2 mRNA表达水平逐渐升高(P<0.01),Nell-1、Ihh、 Ptch-1、 Gli-1和Hhip-1 mRNA表达水平逐渐升高(P<0.01),且在2 000μstrain/2 h时达到最高,随后出现回落但仍明显高于对照组(P<0.01)。Western blotting检测,各组细胞中Nell-1、Runx2和Ihh蛋白表达水平与mRNA表达水平变化趋势一致。环巴胺预处理后,与对照组比较,环巴胺组细胞中Ihh、Ptch-1、Gli-1和Hhip-1 mRNA表达水平均明显降低(P<0.01),�Objective:To discuss the regulatory effect of physiological tensile stress on the differentiation of chondrocytes,and to clarify the associated signaling pathway mechanism.Methods:The ATDC5 chondrocytes were cultured in vitro and subjected to physiological tensile stress by four-point bending cell mechanical loading device.Initially,the cells were divided into control group and tensile stress group(2000μstrain/2 h group),and further divided into different stress magnitudes(1000,2000,and 3000μstrain)for 2 h,and 2000μstrain for different duration time(1,2,and 4 h)groups;the cells without tensile stress were used as control group.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of typeⅡcollagen(Col-Ⅱ),typeⅩcollagen(Col-Ⅹ),aggregated proteoglycom(Aggrecan),sex-determining region Y-box protein 9(SOX9),vascular endothelial growth factor(VEGF),proliferating cell nuclear antigen(PCNA),Nel-like molecule tyep 1(Nell-1),Runt-related transcription factor 2(Runx2),Indian hedgehog(Ihh),patched homolog 1(Ptch-1),GLI family zinc finger protein 1(Gli-1),and hedgehog interacting protein 1(Hhip-1)mRNA in the cells in various groups;Western blotting method was used to detect the expression levels of Nell-1,Runx2,and Ihh proteins in the cells in various groups.The ATDC5 cells were divided into control group,cyclopamine group,tensile stress group,and cyclopamine+tensile stress group.RT-qPCR method was used to detect the expression levels of Nell-1,Ihh,Ptch-1,Gli-1,and Hhip-1 mRNA in the cells in various groups;Western blotting method was used to detect the expression levels of Nell-1 and Ihh proteins in the cells in various groups.Results:Compared with control group,the expression levels of Col-Ⅱ,Col-Ⅹ,Aggrecan,SOX9,VEGF,and PCNA mRNA in the cells in 2000μstrain/2 h group were significantly increased(P<0.01);after treated with 2000μstrain tensile stress for different duration time(1,2,and 4 h)or different tensile stresses(1000,2000,and 3000μstrain)for 2 h,compared with c

关 键 词：生理性拉应力 软骨细胞 Nel样1型分子 印度刺猬因子信号通路 Runt相关转录因子2 

分 类 号：Q254[生物学—细胞生物学]