三种大鼠椎间盘细胞提取、鉴定及在单层和微团培养中的基质表达  

Extraction and characterization of three types of primary cells from rat intervertebral disc and their matrix expression in monolayer and micromass culture

作　　者：胡思远 陈建权 Hu Siyuan;Chen Jianquan(Orthopedic Institute,Suzhou Medical College,Soochow University,Suzhou 215031,Jiangsu Province,China)

机构地区：[1]苏州大学苏州医学院骨科研究所,江苏省苏州市215031

出　　处：《中国组织工程研究》2025年第12期2528-2535,共8页Chinese Journal of Tissue Engineering Research

基　　金：江苏高校优势学科建设工程资助项目(PAPD),项目参与人:陈建权。

摘　　要：背景:目前获得椎间盘原代细胞的方法大多较繁琐,且缺少同时提取纤维环、终板与髓核原代细胞的相关报道,因此找到同时提取3种细胞的方法十分关键。目的:探索一种同时提取并培养大鼠来源3种椎间盘细胞(纤维环、终板与髓核)的方法,对之进行鉴定并探究单层与微团培养对细胞外基质的影响。方法:取材自3周龄雄性SD大鼠椎间盘组织,从其中分离出软骨终板、髓核与纤维环组织。针对髓核及纤维环组织,先使用0.1%链酶蛋白酶E于37℃消化30 min,然后使用0.2%Ⅱ型胶原酶消化4 h来释放其中的细胞;针对终板组织,直接使用0.2%Ⅱ型胶原酶消化4 h来释放其中的细胞。将去除消化酶后的细胞接种于含有培养基的培养皿中,并观察细胞形态;通过实时定量荧光PCR与蛋白印迹实验检测各细胞标记物的表达水平;单层或微团培养大鼠原代纤维环细胞与软骨终板细胞,并采用阿尔新蓝与番红染色检测细胞外基质的表达能力。结果与结论:①3种椎间盘细胞均在培养4 d后开始贴附在皿底,并逐渐展现出增殖的活力;培养至第8天时,3种细胞增殖显著,形态均呈梭形,其中髓核细胞中存在多囊泡的脊索样细胞;②通过实时定量荧光PCR和/或蛋白免疫印迹实验发现原代髓核细胞中K19与Car3表达较高,原代纤维环细胞中Sparc与Bgn表达较高,原代软骨终板细胞中Pth1r与Lars2表达较高;③原代纤维环、软骨终板细胞微团培养后,经阿尔新蓝与番红染色证明这种培养方式相较于单层培养能够提高细胞外基质的表达能力;④结果表明,通过此次实验方法提取并培养获得的原代椎间盘细胞形态较好并具有较高的胞外基质表达水平,这一成果有望为科研人员提供一种新的科研工具,帮助科研人员更好地理解椎间盘生物学。BACKGROUND:Currently,methods for obtaining primary intervertebral disc cells are mostly cumbersome and there is a lack of relevant reports on the simultaneous extraction of three types of cells.Therefore,it is crucial to find a method to simultaneously extract three types of cells.OBJECTIVE:To explore a method for simultaneously extracting and culturing three types of intervertebral disc cells from rats,to identify them,and to investigate the effects of monolayer versus micromass cultures on the extracellular matrix.METHODS:The cartilaginous endplate(CEP),nucleus pulposus(NP),and annulus fibrosus(AF)tissues were separated from 3-week-old male Sprague-Dawley rat intervertebral disc tissue.For the nucleus pulposus and annulus fibrosus tissues,0.1%pronase E was used for 30 minutes of digestion at 37°C followed by 0.2%collagenase type II digestion for 4 hours to release the cells;for the cartilaginous endplate tissue,direct digestion with 0.2%collagenase type II for 4 hours was performed to release the cells.The cells,after removal of the digestion enzymes,were seeded into culture dishes containing culture medium,and their morphology was observed.Real-time fluorescence quantitative PCR and western blot assay were performed to detect the expression levels of various cell markers.Rat primary annulus fibrosus cells and cartilaginous endplate cells were cultured in monolayer or micromass cultures,and Alcian blue and Safranin O staining were used to assess the extracellular matrix expression capability.RESULTS AND CONCLUSION:After 4 days of culture,all three types of intervertebral disc cells began to adhere to the bottom of the dish and gradually showed proliferative vitality.By the 8th day of culture,significant proliferation of the three types of cells was observed,with a spindle-shaped morphology.Notably,the nucleus pulposus cells exhibited multivesicular notochord-like cells.Through real-time fluorescence quantitative PCR and/or western blot assay,it was found that primary nucleus pulposus cells highly expressed Cyt

关 键 词：原代髓核细胞 原代纤维环细胞 原代终板细胞 细胞提取 细胞鉴定 微团培养 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R683