改良酶消化法体外培养、纯化分离人颞下颌关节滑膜A型细胞  

Culture,purification and isolation of human temporomandibular joint synovial type A cells by modified enzyme digestion in vitro

作　　者：万成 黄正 和慧 宗菲 雷印涛 王克强 张婷婷 WAN Cheng;HUANG Zheng;HE Hui;ZONG Fei;LEI Yintao;WANG Keqiang;ZHANG Tingting(Department of Stomatology,Second Affiliated Hospital of Shandong First Medical University,Taian 271000,China;Transformation of Scientific and Technological Achievements,Shandong First Medical University(Shandong Academy of Medical Sciences),Jinan 250117,China;Laboratory of the Second Affiliated Hospital of Shandong First Medical University,Taian 271000,China)

机构地区：[1]山东第一医科大学第二附属医院口腔科,山东泰安271000 [2]山东第一医科大学(山东省医学科学院)成果转化与产业部,山东济南250117 [3]山东第一医科大学第二附属医院检验科,山东泰安271000

出　　处：《山东第一医科大学（山东省医学科学院）学报》2024年第5期257-264,共8页Journal of Shandong First Medical University ＆ Shandong Academy of Medical Sciences

基　　金：泰安市科技创新发展计划(2021NS336)。

摘　　要：目的探讨获取人颞下颌关节滑膜A型细胞的较佳方法,为A型细胞的进一步研究奠定基础。方法分别用组织块法、传统酶消化法与改良的酶消化法培养滑膜细胞,观察培养3 d、7 d、2周、4周时细胞生长情况,记录首次传代时间。通过免疫组化检测衬里层细胞CD68、组织蛋白酶S的表达来检测A型细胞吞噬功能。结果滑膜组织块法接种约7 d后部分组织块周围有细胞游离出来,约4周后滑膜细胞长势接近80%融合密度,可进行首次细胞传代。传统酶消化法细胞贴壁缓慢,4周时才能观察部分细胞团形成,向周围呈放射状生长,约6周后才可首次传代。改良酶消化法接种3 d后,光镜下观察部分单个细胞贴壁,有一些胶原酶未消化完全而又能经过研磨透过滤网的微小组织块或细胞团块,在贴壁后迅速向周围增殖,约2周后可首次传代,3周后再次传代的细胞量与组织块法4周后首次细胞传代时间相同。免疫组化检测CD68有阳性表达,细胞位于靠近关节腔侧,组织蛋白酶S未见阳性表达。结论改良的酶消化法培养对比组织块法、传统酶消化法,在获得相同细胞量的基础上,缩短了原代细胞培养时间。改良酶消化法传代时A型细胞尚在存活期间,给后期A型细胞纯化分离奠定了基础。巨噬细胞标志物Cathepsin S在A型细胞上无表达,提示A型细胞由于体内局部环境的作用,已有别于巨噬细胞。Objective:To explore a better method to obtain human temporomandibular joint synovial type A cells,and lay a methodological foundation for the further study of type A cells.Methods:Synovial cells are cultured with tissue block method,traditional enzyme digestion method and improved enzyme digestion method respectively.The growth of synovial cells is observed at 3,7,2 week and 4 week of culture,and the first passage time is recorded.The expression of CD68 and cathepsin S in lining cells is detected by immunohistochemistry to determine the phagocytic function of type A cells.Results:About 7 days after inoculation with synovial tissue block method,some cells are free around the tissue block,and the growth of synovial cells is close to 80%of the fusion density after about 4 week,which can be used for the first cell passage.The cells adhere slowly with traditional enzyme digestion method,and the formation of some cell clusters can be observed only after 4 week,and grow radially around,and the first passage can be made after about 6 week.Three days after inoculation with the modified enzyme digestion method,we observe under the light microscope that part of the single cells adhere to the wall.There are some small tissue blocks or cell blocks that are not fully digested by collagenase and can be ground through the filter screen.After adhering to the wall,they proliferate rapidly to the surrounding area.After about 2 week,they can be subcultured firstly.After 3 week,the number of cells subcultured is the same as that of the first cell subcultured after 4 week of the tissue block method.Immunohistochemical examination has shown that CD68 is positive,and the cells are located near the articular cavity,while cathepsin S is not positive.Conclusion:Compared with tissue block method and traditional enzyme digestion method,the modified enzyme digestion method can shorten the time of primary cell culture on the basis of obtaining the same cell quantity.Type A cells are still alive during the passage of the modified enzyme digest

关 键 词：滑膜 细胞培养 组织块法 酶消化法 免疫组化 

分 类 号：R782.6[医药卫生—口腔医学]