应用流式-荧光原位杂交协助鉴别诊断EB病毒感染的淋巴细胞亚群  

Application of Flow Cytometry Combined Fluorescence in Situ Hybridization to Indentify the Lymphocyte Subtypies with Epstein-Barr Virus Infection

作　　者：苏虹宇 舒逸[1] 傅国 柳梓杨 朱丹 曾腊梅 马德禹 邹琳[1,2] SU Hong-Yu;SHU Yi;FU Guo;LIU Zi-Yang;ZHU Dan;ZENG La-Mei;MA De-Yu;ZOU Lin(Department of Clinical Molecular Medicine of Children's Hospital in Chongqing Medical University,National Clinical Research Center for Child Health and Disorders,Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing Key Laboratory of Pediatrics,Chongqing 400014,China;Clinical Research Unit of Children's Hospital in Shanghai Jiaotong University,Institute of Pediatric Infection,Immunity,and Critical Care Medicine,Shanghai Jiao Tong University School of Medicine,Shanghai 200062,China)

机构地区：[1]重庆医科大学附属儿童医院临床分子医学中心,国家儿童健康与疾病临床医学研究中心,儿童发育疾病研究教育部重点实验室,儿科学重庆市重点实验室,重庆400014 [2]上海交通大学附属儿童医院临床研究中心,上海交通大学医学院儿童感染免疫与重症医学研究院,上海200062

出　　处：《中国实验血液学杂志》2022年第3期897-907,共11页Journal of Experimental Hematology

基　　金：国家自然科学基金(81870126,82070167,81900190)。

摘　　要：目的:建立流式细胞术-荧光原位杂交联用技术(Flow-FISH),探讨Flow-FISH技术在临床EB病毒(Epstein-Barr virus)感染性疾病中感染细胞亚型鉴定的临床前诊断价值。方法:通过Ficoll-paque离心分离法收集重庆医科大学附属儿童医院招募的9例EBV感染患者的外周血标本中单个核细胞;通过qRT-PCR检测细胞EBER1及EBER2表达;通过荧光标记抗体对细胞表面标志物进行检测,经固定破膜剂对标记后细胞进行固定及破膜处理后与FISH探针37℃条件下过夜杂交,通过流式细胞仪、荧光显微镜检测细胞状态、表面抗原染色以及FISH探针与靶mRNA的结合。结果:优化固定破膜剂配方,建立0.2%Tween-20 4℃破膜15 min为破膜条件,此条件对细胞状态及表面抗原影响小,不干扰流式细胞术对细胞亚群判定。优化杂交液配方,探明20%甲酰胺和7%硫酸葡聚糖在37℃过夜条件下杂交为最优杂交条件,此条件下细胞形态及流式分群好,探针杂交效果佳。通过使用Flow-FISH技术能特异性区分各EBV;以及EBV;淋巴细胞系。通过Flow-FISH技术,成功鉴定临床外周血标本中的EBV;淋巴细胞亚群。结论:初步建立流式细胞术-荧光原位杂交联用检测EBV感染细胞亚群技术,为其进一步的临床前实验奠定基础。Objective: To establish the technique that take the advantages of flow cytometry combined fluorescence in situ hybridization(Flow-FISH) to identify the Epstein-Barr virus(EBV) infected lymphocyte subtypies in patients′ peripheral blood sample. Methods: Peripheral Blood monocyte from 9 patients with EBV infection enrolled at Children′s Hospital in Chongqing Medical University were isolated by Ficoll-paque centrifugal separation. The expressions of EBER1, EBER2 in cell were detected by qRT-PCR. The surface markers of cell were detected by Flow cytometry after staining with their antibodies. The cell was treated Fix-Permeabilization Buffer before hybridization with fluorescent labeled probe at 37 ℃ overnight. The cell status, surface markers and targeted mRNA are detected by flow cytometry and fluorescence microscope. Results: It was optimized that the Fix-Permeabilization Buffer and recipe with 0.2% Tween-20 were picked out as providing a good cell integrity and high resolution of surface markers. Hybridization with 20% formamide and 7% dextran sulfate at 37 ℃ overnight is the optimal hybridization condition as a good hybridization effect, a detectable cell integrity and a high resolution of cell markers under flow cytometry detection. Finally, upon the established Flow-FISH method, lymphocyte subpopulations of the EBV;cells from cell lines and blood samples of patients were identified successfully. Conclusion: A Flow-FISH technology is established,which can be applied in the identification of EBV infected cell subtypes. This research provides a foundmental for its application in clinical test in EBV+ related proliferative diseases.

关 键 词：流式-荧光原位杂交 爱泼斯坦-巴尔病毒 鉴别诊断 EB病毒编码的小RNA 

分 类 号：R446.6[医药卫生—诊断学]