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

摘要/Abstract

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。

关键词:滑膜,细胞培养,组织块法,酶消化法,免疫组化

Abstract:

ObjectiveTo 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.MethodsSynovial 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.ResultsAbout 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.ConclusionCompared 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 digestion method， which can lay a foundation for the purification and separation of type A cells in the later stage. The macrophage marker cathepsin S is not expressed on type A cells， suggesting that type A cells are different from macrophages due to the effect of local environment in vivo.

Key words:synovial membrane,cell culture,tissue block method,enzyme digestion,immunohistochemistry

万成, 黄正, 和慧, 宗菲, 雷印涛, 王克强, 张婷婷. 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[J]. betway必威登陆网址 (betway.com )学报, 2024, 45(5): 257-264.

Cheng WAN, Zheng HUANG, Hui HE, Fei ZONG, Yintao LEI, Keqiang WANG, Tingting ZHANG. Culture， purification and isolation of human temporomandibular joint synovial type A cells by modified enzyme digestion in vitro[J]. Journal of ShanDong First Medical University&ShanDong Academy of Medical Sciences, 2024, 45(5): 257-264.

图/表4

图1倒置显微镜下滑膜细胞形态（ × 100）A示组织块法培养3 d后周围有极少量细胞爬出；B示组织块法培养7 d后周围细胞爬出增多；C示组织块法培养14 d后细胞爬出明显增多。D示传统酶消化法培养3 d后部分细胞贴壁；E示传统酶消化法培养7 d后贴壁细胞增多；F示传统酶消化法培养14 d后细胞逐渐增殖。G示改良酶消化法3 d后细胞团块周围细胞爬出；H示改良酶消化法培养7 d后微小组织块周围细胞大量爬出；I示改良酶消化法培养14 d后细胞长满3.5 cm培养皿。

图2人颞下颌关节滑膜切片HE染色衬里层结构（ × 40）A示正常滑膜衬里层；B示衬里层显著增厚；C示衬里层稍增厚；D示单层的衬里层结构；E示衬里层细胞明显变形，呈细长梭形；F示大鼠T滑膜衬里层结构。标尺为25 μm。

图3人颞下颌关节滑膜切片滑膜衬里层A型细胞CD68阳性表达（IHC， × 100）A示正常滑膜衬里层的三层细胞结构，CD68阳性的细胞较少，位于靠近关节腔侧的衬里层表层；B示稍增厚的衬里层结构，CD68阳性的细胞较少，位于靠近关节腔侧的衬里层表层；C示单层的衬里层结构，CD68阳性的细胞较少，与无阳性染色的细胞间隔分布；D为肺癌周围正常肺组织切片，其中含大量肺泡巨噬细胞，作为CD68表达的阳性对照，可观察到肺泡巨噬细胞CD68强阳性表达。标尺为40 μm。

图4人颞下颌管家滑膜切片滑膜衬里层A型细胞Cathepsin S 阳性表达（IHC， × 100）A、B、C为人颞下颌关节滑膜切片，滑膜衬里层细胞组织蛋白酶S均无阳性表达；D为肺癌周围正常肺组织切片，其中含大量肺泡巨噬细胞，作为组织蛋白酶S表达的阳性对照，可观察到肺泡巨噬细胞Cathepsin S阳性表达。标尺为40 μm。

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