心肌纤维化特殊染色方法比较与优化  

Comparison and optimization of special staining methods forobservation of myocardial fibrosis

作　　者：王亚恒 马嘉昕 雷雨 张连峰 吕丹 WANG Yaheng;MA Jiaxin;LEI Yu;ZHANG Lianfeng;LYU Dan(Key Laboratory of Comparative Medicine,National Health Commission of China(NHC),Institute of Laboratory Animal Science,Chinese Academy of Medical Sciences,Peking Union Medical College,Beijing 100021,China;Beijing Engineering Research Center for Experimental Animal Models of Human Critical Diseases,Institute of Laboratory Animal Science,Chinese Academy of Medical Sciences,Peking Union Medicine College,Beijing 100021,China;National Center of Technology Innovation for animal model,Institute of Laboratory Animal Science,Chinese Academy of Medical Sciences,Peking Union Medicine College,Beijing 100021,China;National Human Diseases Animal Model Resource Center,Institute of Laboratory Animal Science,Chinese Academy of Medical Sciences,Peking Union Medicine College,Beijing 100021,China)

机构地区：[1]中国医学科学院医学实验动物研究所,国家卫生健康委比较医学重点实验室,北京100021 [2]中国医学科学院医学实验动物研究所,北京市人类重大疾病实验动物模型工程技术研究中心,北京100021 [3]中国医学科学院医学实验动物研究所,国家人类疾病动物模型资源库,北京100021 [4]中国医学科学院医学实验动物研究所,国家动物模型技术创新中心,北京100021

出　　处：《中国实验动物学报》2024年第3期347-354,共8页Acta Laboratorium Animalis Scientia Sinica

基　　金：中国医学科学院医学与健康科技创新工程(2022-I2M-1-020);中央高校基本科研业务费(3332023054)。

摘　　要：目的 对目前已有心肌纤维化染色方法进行优化,弥补目前心肌纤维常用染色方法在定量分析中存在的胶原纤维漏读及误读等问题,为心肌纤维化半定量及诊断提供参考。方法 利用实验室前期构建的心肌组织特异性cTnT~(R141W)基因突变致扩张型心肌病转基因小鼠模型,制备心脏组织石蜡切片,分别进行马松三色(Masson’s trichrome, Masson)染色法、天狼星红(picric sirius red, PSR)染色法、天狼星红-品红(van Gieson, VG)染色法、天狼星红-固绿(sirius red/fast green staining, SR/FG)染色法染色,利用Image J 2.1.0软件对胶原纤维面积进行定量比较,并从染液浓度、染色时间、酸性溶液预染3个方面对SR/FG染色法进行优化,对后续的胶原纤维定量分析进行应用验证。结果 4种特染方法均可实现胶原纤维的分布观察,其中,优化后的SR/FG染色法,即0.1%天狼星红-苦味酸酸性溶液预染5 min,随后于0.1%天狼星红-苦味酸与0.04%固绿的混合液中染色1 h,在软件识别中具有更低的漏读率和遗失率。结论 本文优化后的SR/FG染色法较其他传统胶原纤维特染方法,胶原纤维与心肌组织着色鲜亮、颜色对比明显、稳定性佳、方便快捷,更适用于后续胶原纤维占比定量分析应用。Objective The existing dyeing methods of myocardial fibrosis were optimized to make up for the problems of missing and misreading of collagen fibers in the quantitative analysis of the current common dyeing methods of myocardial fibers,and to provide a reference for the semi\|quantitative and diagnosis of myocardial fibrosis.Methods Paraffin sections of cardiac tissue were prepared using a transgenic mouse model of cardiomyopathy with a specific laboratory-constructed cTnT R141W gene mutation.Four staining method were performed for comparative observations:Masson’s trichrome(Masson)staining,picrosirius red(PSR)staining,van Gieson(VG)staining,and Sirius red/fast green(SR/FG)staining.Image J 2.1.0 software was used to quantitatively compare the areas of collagen fibers.SR/FG was optimized from three aspects:dye concentration,staining time,and acid solution prestaining,and the quantitative analysis of collagen fibers was then verified.Results The collagen fiber distribution was observed by the four staining method,among which SR/FG was notable.It involved prestaining with a 0.1%Sirius red-picric acid acidic solution for 5 min,adjusting the concentration of the dye solution to 0.1%Sirius red-picric acid and 0.04%fast green mixture,and incubating the sections in the mixed staining solution for 1 h.This method exhibited the lowest incidence of missed readings and loss in determining the proportion of collagen fibers.Conclusions Compared with other traditional collagen fiber staining method,the optimized SR/FG technique described in this paper produces bright coloring of collagen fibers and myocardial tissue,obvious color contrast,and high stability,convenience,and speed.It is suitable for subsequent quantitative analysis and determination of the collagen fiber proportion.

关 键 词：心肌纤维化 特殊染色方法 天狼星红-固绿 定量分析 

分 类 号：Q95-33[生物学—动物学]