一种用于肌腱胶原纤维束及周围腱内膜组分分布形态学观察的快速组织学制片方法研究  

A rapid histological preparation method for observation of morphology and composition distribution of tendon collagen fascicle and endotendinium

作　　者：张忆[1] 周利 唐林巧 秦廷武[2] 宁良菊[2] ZHANG Yi;ZHOU Li;TANG Linqiao;QIN Tingwu;NING Liangju(Research Core Facility of West China Hospital,Sichuan University,Chengdu Sichuan,610041,P.R.China;Laboratory of Stem Cell and Tissue Engineering,West China Hospital,Sichuan University,Chengdu Sichuan,610041,P.R.China)

机构地区：[1]四川大学华西医院公共实验技术中心,成都610041 [2]四川大学华西医院干细胞与组织工程研究室,成都610041

出　　处：《中国修复重建外科杂志》2019年第9期1169-1173,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(31600783、31870968);四川省重点研发项目(2018SZ0044、2018SZ0052)~~

摘　　要：目的探讨一种用于观察肌腱胶原纤维束及周围腱内膜形态与组分分布的快速组织学制片方法。方法以猪趾浅屈肌腱组织为例,行冰冻切片,片厚6μm。大体观察切片完整性,HE染色观察组织完整性与冰晶形成情况。然后,将切片分为5组,A组行过碘酸雪夫(Priodic acid-Shiff,PAS)染色,B组行Masson染色,C组行网状纤维染色,D组行Ⅲ型胶原免疫组织化学和免疫荧光染色,E组切片经65℃烘烤10 min后行Masson染色。观察各组肌腱胶原纤维束与周围腱内膜组分分布情况。结果大体观察切片连续均匀;HE染色见组织整体结构完整清晰,无冰晶空隙形成,但无法观察到胶原纤维束与周围腱内膜组分分布情况。A、B、C组肌腱组织整体单一染色,无法辨认胶原纤维束与周围腱内膜组分分布;D组周围腱内膜单独弱显色,两组分能差异化染色,但对比度不明显;E组胶原纤维束与周围腱内膜差异化染色,两组分清晰可见。结论猪趾浅屈肌腱冰冻切片后,通过65℃烘烤10 min联合Masson染色方法可以快速准确显示胶原纤维束与周围腱内膜组分分布形态学表征。Objective To explore a rapid histological preparation method to observe morphology and composition distribution of tendon collagen fascicle and endotendinum. Methods Taking porcine superflexor tendon of foot as an example, tendons were sliced into sections with 6 μm by frozen section technology, after which general observation of the section integrity was carried out. After fixed with 10% neutral buffered formalin and performed with HE staining, the tissue integrity and ice crystal formation were observed under microscope. Sections were then divided into 5 groups by different methods of dyeing. Group A: Priodic acid-Shiff(PAS) staining;group B: Masson staining;group C: reticular fibers staining;group D: immunohistochemical and immunofluorescent staining of type Ⅲ collagen;group E:the sections were baked at 65℃ for 10 minutes and stained with Masson. The composition distribution of tendon collagen fascicle and endotendinum in different groups were observed. Results From general observation, the frozen section of tendon tissue was complete and continuous. Although the tissue integrity in the tendon sections could be seen and no ice crystal was formed, the composition distribution could not be identified by HE staining. The entire tendons in groups A,B, and C were dyed, and the composition distribution of collagen fascicle and endotendinum could not be identified. The endotendinum in group D was stained weakly positive for type Ⅲ collagen alone, and the two components were differentiated dyed but the contrast was not obvious. In group E, the collagen fascicle and endotendinium were differentiated dyed and the two components in tendon tissue were clearly visible. Conclusion The morphology and the composition distribution of tendon collagen fascicle and endotendinum can be characterized rapidly and accurately, using a combination of baking at 65℃ for 10 minutes and Masson staining after porcine superflexor tendons were sliced by frozen section technology.

关 键 词：肌腱 冰冻切片 烘片 MASSON染色 

分 类 号：R361.2[医药卫生—病理学]