摘要
背景:常用的获得软骨细胞的方法有机械-酶消化法、链霉蛋白酶和胶原酶序贯消化法、胰蛋白酶和Ⅱ型胶原蛋白酶序贯消化法以及单纯Ⅱ型胶原蛋白酶消化法。目的:探讨SD大鼠膝关节软骨细胞Ⅱ型胶原酶体外分离、提取、鉴定方法,观察原代至第3代软骨细胞的形态学特点。方法:课题组采用一步酶消化法(Ⅱ型胶原酶)体外分离、提取、培养SD大鼠膝关节软骨细胞并进行传代培养,进而构建其体外培养体系。使用细胞增殖实验、倒置相差显微镜观察细胞组织学形态;甲苯胺蓝染色、Ⅱ型胶原与蛋白聚糖免疫荧光染色等方法进行细胞性状鉴定。实验经大连大学附属中山医院伦理委员会批准。结果与结论:①镜下观察与细胞增殖实验均显示第2,3代细胞形态、增殖能力最佳,后逐渐退化;②甲苯胺蓝染色、Ⅱ型胶原与蛋白聚糖免疫荧光染色等组织学检测显示,第3代体外培养软骨细胞状态功能良好;③结果表明,一步酶消化法(Ⅱ型胶原酶)能够成功地从SD大鼠膝关节标本中分离出软骨细胞并且可以进行传代培养;体外培养3代后软骨细胞去分化特性逐渐增强,第3代体外培养软骨细胞性状良好,可用于实验研究。
BACKGROUND:Common methods for obtaining chondrocytes include mechanical-enzymatic digestion,sequential digestion of pronase and collagenase,sequential digestion of trypsin and type II collagenase,and simple type II collagenase digestion.OBJECTIVE:To investigate the isolation,extraction and identification of knee joint chondrocytes from Sprague-Dawley rats,and to observe the morphological characteristics of chondrocytes from primary to passages 3.METHODS:We used single-step digestive approach(type II collagenase digestion)to isolate and obtain chondrocytes from the knee joints of SpragueDawley rats in vitro,and then established a culture system in vitro.Cell proliferation assay and inverted phase contrast microscope were used to observe morphohistology of the cells.Toluidine blue staining,type II collagen as well as proteoglycan immunofluorescence staining were used to identify the characteristics of chondrocytes.The experimental protocol was approved by the Ethics Committee of Zhongshan Hospital of Dalian University.RESULTS AND CONCLUSION:Microscopic observation and cell proliferation assay both showed satisfactory cell morphology and proliferation at passages 2-3,and the capacity of the cells decreased gradually.Moreover,toluidine blue staining,type II collagen and proteoglycan immunofluorescence staining revealed superior characteristics of passage 3 chondrocytes cultured in vitro.These findings indicate that chondrocytes can successfully be isolated by the singlestep type II collagenase digestion.Cell proliferation and passage cultivation can also be achieved.And dedifferentiation capacity of chondrocytes is improved gradually after the passages 3 cultured in vitro.Passage 3 cells cultured in vitro have superior characteristics for the use in experimental researches.
作者
杨帆
刘保一
刘家河
杨佳慧
覃开蓉
赵德伟
Yang Fan;Liu Baoyi;Liu Jiahe;Yang Jiahui;Qin Kairong;Zhao Dewei(School of Biomedical Engineering,Dalian University of Technology,Dalian 116024,Liaoning Province,China;Department of Orthopedics,Zhongshan Hospital,Dalian University,Dalian 116001,Liaoning Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2021年第14期2161-2165,共5页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金委项目(81672139,81371942),项目负责人:赵德伟
国家重点研发计划(2016YFC1102000),项目负责人:赵德伟
大连市科技创新基金项目(2018J11CY030),项目负责人:赵德伟。
关键词
软骨细胞
酶消化法
细胞培养
大鼠
实验
chondrocyte
enzyme digestion
cell culture
rat
experiment
