冰冻切片法观察不脱钙骨组织的荧光分布

FLUORESCENCE DISTRIBU TION IN BONE AND CARTIL AGE TISSUE BY SECTIONING OF FROZEN UNDECALCIFIED BONE

目的介绍一种不脱钙骨组织的冰冻切片法,通过观察骨和软骨内的荧光分布,探讨该方法的可行性。方法取健康成年雄性绿色荧光蛋白转基因SD大鼠膝关节,经包埋剂包埋,置于冰冻切片机中,将表面涂有合成橡胶的薄膜黏附于包埋块表面,行膝关节组织切片。荧光显微镜及光镜下观察切片组织内荧光表达及结构;并行Ⅰ、Ⅱ型胶原免疫荧光染色和HE、甲苯胺蓝、茜素红染色,观察软骨及骨的结构及荧光分布特征。结果制备的大鼠膝关节冰冻切片厚度均为6μm,大体观察示关节各结构均完整切割,光镜下能清晰分辨切片软骨各层细胞形态以及软骨下骨、骨小梁排列走行。荧光显微镜下观察,可见关节周围软组织、软骨及骨组织呈绿色荧光,Ⅰ型胶原在骨组织表达,Ⅱ型胶原在软骨组织表达。HE染色及甲苯胺蓝染色能清晰分辨软骨层形态结构。茜素红染色示软骨下骨板、半月板内部组织结构完整,而且胫骨近端的皮质骨连续性存在。结论采用冰冻切片法制备不脱钙骨组织切片,可直接观察骨、软骨组织内荧光物质的分布,缩短骨组织切片的制备周期。

Objective To introduce a technique of frozen sections for undecalcified bone and discuss its feasibility by observing the fluorescence distribution of the bone and cartilage. Methods The male Sprague Dawley transgenic rats at the age of 8 weeks, which express green fluorescent protein were selected to isolate the whole knee sectioned by the undecalcified bone frozen section technique. Under the fluorescence and light microscopy, the fluorescence and structure were observed within the organization of slice. Immunohistochemical staining（collagen type I and II）, HE staining, toluidine blue staining, and Alizarin red staining were performed to observe the distribution of fluorescent substance and cartilage and bone structure. Results The thickness of sections prepared by this technology was 6 μm. General observation showed that the structure of sectioned joint was complete. Under the light microscope, the morphology of cartilage cells, the arrangement of subchondral bone, and trabecular bone traveling could be clearly distinguished. Under fluorescence microscope, green fluorescence was shown in the joint soft tissue, cartilage tissue, and bone tissue; collagen type I expressed in the bone tissue, collagen type II in cartilage tissue. HE staining and toluidine blue staining could clearly distinguish the morphology of the cartilage layer. Alizarin red staining showed the structural integrity of subchondral bone plate and the organization within the meniscus, and proximal tibia cortical bone continuity. Conclusion The fluorescence distribution can be directly observe in the bone and cartilage by sectioning of frozen undecalcified bone. This new technology can shorten the cycle of preparing sections.