一种用于肌腱二级纤维束及周围腱内膜形态观察的扫描电镜制样方法研究  

A Sampling Method for Scanning Electron Microscopic Observation of Morphology of Tendon Secondary Fiber Bundle and Endotendinium

作　　者：张忆[1] 苏波[1] 杨桢[1] 胡若楠 宁良菊[2] ZHANG Yi;SU Bo;YANG Zhen;HU Ruonan;NING Liangju(Research Core Facility,West China Hospital,Sichuan University,Chengdu 610041,China;Laboratory of Stem Cell and Tissue Engineering,West China Hospital,Sichuan University,Chengdu 610041,China)

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

出　　处：《组织工程与重建外科杂志》2020年第1期34-38,共5页Journal of Tissue Engineering 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：目的探讨一种用于肌腱二级纤维束及周围腱内膜形态观察的扫描电镜制样方法。方法以猪趾浅屈肌腱为例,将肌腱在常温下横切为1 cm左右的肌腱段,通过双光子荧光显微镜观察组织完整性,行冰冻切片联合特殊Masson染色,观察肌腱二级纤维束及周围腱内膜分布情况。另取肌腱段若干经10%中性甲醛固定4 h,分为5组,采用不同的扫面电镜制样方法:A组,单纯梯度脱水;B组,行冰冻切片,再将肌腱切片进行梯度脱水;C组,梯度脱水至70%,行冰冻切片,再将肌腱切片进行梯度脱水;D组,梯度脱水至80%,行冰冻切片,再将肌腱切片进行梯度脱水;E组,梯度脱水至100%,行冰冻切片。各梯度脱水试剂为乙醇配制,梯度脱水终浓度均为100%。冰冻切片厚度均为100μm。经临界点干燥喷金后行体视显微镜和扫描电镜观察。结果双光子二次谐波显微成像观察示肌腱结构完整连续。Masson染色观察示周围腱内膜较薄区域难以辨别独立胶原纤维束。体视显微镜观察示肌腱切片电镜制样后大体完整,三级纤维束的周围腱内膜清晰可见。扫描电镜观察示A、D、E组肌腱组织呈现不同程度的过度收缩,二级纤维束结构无法辨别;B组整体呈现无规律的冰晶空隙,二级纤维束结构难以辨认,胶原纤维排列清晰可见;C组连续完整,二级纤维束及其周围腱内膜、胶原纤维排列均清晰可见。结论扫描电镜的肌腱试样制备,可通过组织固定后经乙醇梯度脱水至70%,行冰冻切片,再将肌腱切片继续梯度脱水至100%,该方法能清晰观察到肌腱二级纤维束及周围腱内膜的形态。Objective To explore a sampling method for scanning electron microscopic observation of morphology of tendon secondary fiber bundle and endotendinium.Methods Taking porcine super exor tendon of foot as an example,the tendon was transversely cut into 1 cm segments at room temperature.Tissue integrity was observed by two-photon fluorescence microscopy.Combination of frozen sections and special Masson staining were used to observe the distribution of tendon secondary fiber bundle and endotendinium.Several tendon segments were fixed with 10%neutral formaldehyde for 4 hours and divided into 5 groups.Different sampling methods for scanning electron microscopic observation were used:group A,simple gradient dehydration;group B,frozen section and continued gradient dehydration of tendon slices;group C,gradient dehydration to 70%,frozen section and continued gradient dehydration of tendon slices;group D,gradient dehydration to 80%,frozen section and continued gradient dehydration of tendon slices;group E,gradient dehydration to 100%and frozen section.The gradient dehydration reagents were prepared with ethanol and the final concentration of gradient dehydration was 100%.The thickness of frozen section was 100μm.After critical point drying and gold sputter coating,the specimens were observed by stereo microscope and scanning electron microscope.Results Two-photon second harmonic imaging microscopy showed that the tendon structure was complete and continuous.Masson staining showed that it was difficult to distinguish the independent collagen bundles in the thin area of the endotendinium.Stereo microscopy showed that the tendon slices were grossly intact after sampling for scanning electron microscopy,and the endotendinium around the tertiary fiber bundle was clearly visible.According to scanning electron microscopy,the tendon tissue in group A,D and E showed excessive contraction in varying degrees,and secondary fiber bundle structure could not be distinguished.In group B,the whole tendon tissue showed irregular ice crystal

关 键 词：肌腱 梯度脱水 冰冻切片 扫描电镜 

分 类 号：R329.4[医药卫生—人体解剖和组织胚胎学]