摘要
目的优化原代人Tenon’s囊成纤维细胞(HTFs)体外分离、培养的实验方法,并对体外培养的HTFs的生长特性进行观察。方法采用优化的组织块贴壁法培养原代HTFs;倒置显微镜观察HTFs的基本形态特征;免疫荧光染色Vimentin及Cytokeratin对所培养的原代细胞进行鉴定;采用噻唑蓝比色试验检测细胞增殖活性及生长曲线;流式细胞术检测细胞周期及增值指数。结果组织块贴壁后4~7 d可以见长梭形细胞长出,2~3周后可传代。细胞生长曲线近似“S”形,MTT法显示在第3~5 d细胞增殖能力显著。免疫荧光染色Vimentin阳性率达98%,Cytokeratin阴性,符合HTFs特性且无上皮细胞污染。第3~4代HTFs中(63.48±10.22)%处于G0/G1期,(10.80±5.07)%处于G2/M期,(24.34±8.07)%处于S期,HTFs的增殖指数为35.6%,细胞增殖良好。结论建立了一种简单、经济、有效的获取原代人Tenon’s囊成纤维细胞的方法,为研究青光眼滤过泡瘢痕化提供了可靠的靶细胞模型。
Objective To optimize the experimental method of isolation and culture of primary human Tenon's capsule fibroblasts(HTFs)in vitro,and observe the growth characteristics of HTFs.Methods Primary HTFs were cultured by the optimized adherent method.Morphological characteristics of the HTFs were observed by inverted microscope and the HTFs were identified by Vimentin and Cytokeratin immunofluorescence staining.The cell proliferation activity and growth curve were measured by methyl thiazolyl tetrazolium(MTT)assay,and cell cycle and proliferation index were measured by flow cytometry.Results The long spindle cells could be seen after 4~7 days of the tissues attachment to the petri dish,and passaged after 2~3 weeks.The cell growth curve approximated the"S"shape,and the cell proliferation ability was remarkable at 3-5 days after the cell passage.The positive rate of Vimentin immunofluorescence staining was 98%and that of Cytokeratin immunofluorescence staining was negative,which was consistent with the characteristics of HTFs without epithelial cell contamination.The results of flow cytometry showed that in the 3~4rd generation of HTFs,(63.48±10.22)%cells were in G0/G1 phase,(10.80±5.07)%cells were in G2/M phase,and(24.34±8.07)%cells were in S phase.The proliferation index of HTFs was 35.6%,representing well cell proliferation.Conclusions A simple,economical and effective for culturing and obtaining HTFs was established,which provided a reliable experimental cytological model for the basic research of filtering bleb scar after glaucoma filtration.
作者
王丽君
李宏松
张文怡
廖丁莹
杨熹婷
王建明
Wang Lijun;Li Hongsong;Zhang Wenyi;Liao Dingying;Yang Xiting;Wang Jianming(Department of Ophthalmology,The Second Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710004,China)
出处
《临床眼科杂志》
2020年第6期551-555,共5页
Journal of Clinical Ophthalmology
基金
陕西省重点研发计划(2018ZDXM-SF-045)
西安市科技计划[2017116SF/YX010(10)]。
