氯化钴诱导体外人髓核细胞缺氧模型的建立  

Establishment of anoxia model of human nucleus pulposus cells induced by cobalt chloride in vitro

作　　者：李欣怡 王洪伸 谭傲威 吴浩杰 李永津[2] 陈博来[2] LI Xin-yi;WANG Hong-shen;TAN Ao-wei;WU Hao-jie;LI Yong-jin;CHEN Bo-lai(The Second Clinical Medical College of Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong,China;不详)

机构地区：[1]广州中医药大学第二临床医学院,广东广州510405 [2]广东省中医院骨一科,广东广州510120 [3]广东省中医药科学院,广东广州510120

出　　处：《广东医学》2023年第6期729-734,共6页Guangdong Medical Journal

基　　金：国家自然科学基金面上项目(82174396,82274554);广东省自然科学基金面上项目(2021A1515011474、2021A1515011455);广东省普通高校创新团队项目(2021KCXTD020);广州市科技计划项目(202102010012,202201020324,202201020554);中国博士后科学基金项目(2021M690758);广东省中医药局中医药科研项目(20212068)。

摘　　要：目的观察氯化钴(CoCl_(2))对体外培养的原代髓核细胞的影响,探寻符合研究目的体外化学干预低氧模型。方法利用贴壁法培养原代髓核细胞,使用100μmol/L、200μmol/L、300μmol/L CoCl_(2)溶液分别处理12 h、24 h、36 h、48 h,形成最终13个分组:空白对照组,CoCl_(2)处理组(100μmol/L-12 h组,100μmol/L-24 h组,100μmol/L-36 h组,100μmol/L-48 h组;200μmol/L-12 h组,200μmol/L-24 h组,200μmol/L-36 h组,200μmol/L-48 h组;300μmol/L-12 h组,300μmol/L-24 h组,300μmol/L-36 h组,300μmol/L-48 h组)。利用CCK-8实验观察不同条件下细胞生长、凋亡情况,并通过免疫印迹(western blot)及实时定量聚合酶链反应(qPCR)检测低氧诱导因子-1α(HIF-1α)、血管内皮生长因子(VEGF)的表达情况。结果与空白对照组比较,CoCl_(2)处理组的细胞生存率整体趋势随着浓度升高、时间延长而降低(P<0.05),但在低浓度、短时间的CoCl_(2)诱导下,髓核细胞的生存率反而提高(P<0.05)。HIF-1α与VEGF的mRNA及蛋白表达亦随CoCl_(2)浓度升高和时间延长而增加(P<0.05)。但在300μmol/L CoCl_(2)的诱导下,HIF-1α、VEGF蛋白的表达进入平台期。结论CoCl_(2)对原代髓核细胞的增殖、凋亡及相关基因、蛋白的表达与时间和浓度相关,总体成逐渐升高趋势。本文成功建立起一种体外培养原代髓核细胞缺氧诱导方法,可以作为研究椎间盘髓核细胞缺氧模型的参考。Objective To observe the effect of cobalt chloride(CoCl_(2))on primary nucleus pulposus cells cultured in vitro,and to explore a chemical intervention model for hypoxia in vitro.Methods Primary nucleus pulposus cells were cultured by adherent method and treated with 100μmol/L,200μmol/L and 300μmol/L CoCl_(2) solution for 12 h,24 h,36 h and 48 h,respectively.The cell growth and apoptosis were observed by CCK-8 assay under different conditions.The expressions of hypoxia-inducing factor 1α(HIF-1α)and vascular endothelial growth factor(VEGF)were assessed by Western Blot and quantitative real-time PCR.Results Compared with blank control group,the overall trend of cell survival rate in CoCl_(2) treatment group was significantly reduced with the increase of concentration and time(P<0.05),but the survival rate of nucleus pulposus cells was significantly increased under the induction of low concentration and short time of CoCl_(2)(P<0.05).The mRNA and protein expression levels of HIF-1αand VEGF were also significantly increased with the increase of CoCl_(2) concentration and time(P<0.05).However,the expression of HIF-1αand VEGF reached a plateau under the induction of 300μmol/L CoCl_(2).Conclusion The proliferation,apoptosis,and expression of related genes and proteins in primary nucleus pulposus cells are correlated with the time and concentration of CoCl_(2),showing an overall increasing trend.This study successfully established an in vitro culture method for inducing hypoxia in primary nucleus pulposus cells,which can serve as a reference for studying the hypoxia model of intervertebral disc nucleus pulposus cells.

关 键 词：氯化钴 椎间盘 髓核细胞 缺氧模型 缺氧诱导因子-1α 血管内皮生长因子 

分 类 号：R681.53[医药卫生—骨科学] N33[医药卫生—外科学]