TSA法多重免疫荧光染色在猴艾滋病模型病理研究中的应用  

Application of multiple immunofluorescence staining based on TSA method in pathological study of simian acquired immunodeficiency disease syndrome(SAIDS)model

作　　者：何晓妍 郑宏毅 郑永唐[1,2] HE Xiao-yan;ZHENG Hong-yi;ZHENG Yong-tang(Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences,Kunming Institute of Zoology,Chinese Academy of Sciences,Kunming,Yunnan 650223;University of the Chinese Academy of Sciences,Beijing 100049)

机构地区：[1]中国科学院昆明动物研究所,中国科学院动物模型与人类疾病机理重点实验室,云南昆明650223 [2]中国科学院大学,北京100049

出　　处：《赣南医学院学报》2021年第8期768-776,共9页JOURNAL OF GANNAN MEDICAL UNIVERSITY

基　　金：国家自然科学基金资助项目(81771770,82071847);NSFC-云南联合基金资助项目(U1802284)。

摘　　要：目的:建立适合石蜡切片的多重免疫荧光染色方法和配套的定量分析算法,用以研究猕猴艾滋病(SAIDS)模型的免疫病理特征。方法:选取SIV感染猕猴和健康猕猴淋巴结的石蜡切片,以优化后的酪胺信号放大技术(TSA)进行荧光染色,通过几轮微波加热洗去前次标记抗体的方式,达到多重染色的目的。对显微图像进行粒度和灰度分析,计算每一个细胞的位置和目的蛋白的表达特征。最后图像信息被转化成数字矩阵,经R语言编程处理后用于定量分析。结果:TSA法经过优化后,切片的荧光背景信号降低而信噪比增加。荧光图像的定量分析表明,SAIDS快进展者淋巴结的CD4^(+)细胞的感染更为严重(P<0.0001),过度浸润滤泡的CD8^(+)细胞异常高表达颗粒酶B(P<0.0001)。结论:本研究建立的TSA法多重免疫荧光染色和配套的定量分析方法是提升病理研究内容的良好技术手段,有助于AIDS及其他疾病发病机制的研究。Objective:Establishing a multiple immunofluorescence staining method suitable for paraffin sections and a supporting quantitative analysis algorithm to study of simian acquired immunodeficiency disease syndrome(SAIDS)model.Methods:The paraffin sections of lymph nodes from SIV-infected and healthy rhesus macaques were selected for fluorescent staining with optimized tyramine signal amplification technology(TSA).Through several rounds of microwave heating to wash away the previously labeled antibody,the purpose of multiple staining is achieved.Analyze the microscopic images with granularity and gray scale,and calculate the location and the expression characteristics of target proteins of each cell.Finally,the image information is converted into a digital matrix,which is processed by R and used for quantitative analysis.Results:After the optimization of the TSA method,the fluorescent background signal decreases and the signal-to-noise ratio increases.Quantitative analysis of fluorescence images showed that in lymph nodes of animals with rapid progression,CD4+cells are more severely infected(P<0.0001),and CD8+cells that over infiltrate follicles abnormally express granzyme B(P<0.0001).Conclusion:Our established multiple immunofluorescence staining based on TSA method and its supporting quantitative analysis methods are good technique to improve the content of pathological research,and is helpful to the study of the pathogenesis of AIDS and other diseases.

关 键 词：TSA HIV AIDS SIV 多重免疫荧光 猕猴动物模型 病理定量分析 

分 类 号：R392.31[医药卫生—免疫学]