体外培养人胸腺组织切片残余细胞类型、分子表达谱与质量评估的研究  

Residual cell types,molecular expression profiles and quality assessment of in vitro cultured human thymic slices

作　　者：关万青 罗贵华 撖菁萱 向群 安云飞[4] 赵录[5] 糜建红 冯泽清 吴玉章 GUAN Wanqing;LUO Guihua;HAN Jingxuan;XIANG Qun;AN Yunfei;ZHAO Lu;MI Jianhong;FENG Zeqing;WU Yuzhang(Department of Immunology,School of Basic Medical Sciences,Southern Medical University,Guangzhou,Guangdong;Chongqing International Institute for Immunology,Chongqing;School of Pharmacy and Bioengineering,Chongqing University of Technology,Chongqing;Department of Rheumatology and Immunology,Chongqing;Department of Cardiothoracic Surgery,Children’s Hospital Affiliated to Chongqing Medical University,Chongqing;Department of Immunology,College of Basic Medical Sciences,Army Medical University(Third Military Medical University),Chongqing,China)

机构地区：[1]南方医科大学基础医学院免疫学教研室,广东广州 [2]重庆国际免疫研究院,重庆 [3]重庆理工大学药学与生物工程学院,重庆 [4]重庆医科大学附属儿童医院风湿免疫科,重庆 [5]重庆医科大学附属儿童医院心胸外科,重庆 [6]陆军军医大学(第三军医大学)基础医学院免疫学教研室,重庆

出　　处：《陆军军医大学学报》2025年第9期893-903,共11页Journal of Army Medical University

基　　金：重庆市技术创新与应用发展专项重大项目(CSTB2023TIAD-STX0012)。

摘　　要：目的拟通过单细胞转录组测序技术(single-cell RNA sequencing,scRNA-seq)解析移植前体外培养人胸腺组织切片的残余细胞类型和功能,并利用培养上清液分子标志物的水平特征建立胸腺组织切片质量评估方法。方法收集2023年5月至2024年1月在重庆医科大学附属儿童医院心胸外科接受心脏外科手术的18例先天心脏病患者废弃的胸腺制备成胸腺组织切片。体外培养14 d后,通过scRNA-seq鉴定残余的细胞类型,联合基因本体论(gene ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)富集分析残余细胞的功能,并查阅胸腺组织切片培养的相关文献筛选出指示胸腺细胞功能的分子标志物。采用ELISA检测上清液分子标志物的蛋白水平变化,绘制受试者工作特征曲线(receiver operating characteristic curve,ROC),以曲线下面积(area under the curve,AUC)判定上清液分子标志物对胸腺组织切片质量的评估价值。将分子标志物判定为质量检测合格与质量检测不合格的胸腺组织切片移植至6~8周龄Balb/c-nude雄性小鼠(体质量14~17 g)皮下(对照组不移植胸腺组织切片),通过流式细胞术和组织学检测分析移植后免疫重建效果。结果(1)scRNA-seq数据显示,胸腺组织切片中含有11种细胞,主要细胞为上皮细胞、成纤维细胞和T细胞。GO和KEGG富集分析显示,上皮细胞与趋化作用、上皮细胞发育、细胞基质粘附、紧密连接等条目相关;成纤维细胞主要与细胞外基质组织、上皮细胞增殖、负向调控细胞迁移、肌动蛋白细胞骨架调节等条目相关;T细胞主要与T细胞分化、T细胞活化的调控、T细胞凋亡、T细胞受体信号等条目相关。(2)筛选出CCL19、CCL21、CXCL12、CXCL16、IL16、SELL作为指示胸腺细胞功能的分子标志物,与第1天相比,CCL19、CCL21、CXCL12和CXCL16蛋白分泌量伴随体外培养显著增加(P<0.05),IL16和L-selectin(SELL表达分�Objective To analyze the composition and function of residual cells in pretransplantation human thymic slices by single-cell transcriptomics sequencing(scRNA-seq), and established a quality assessment method for thymic slices based on the expression levels of molecular markers in the culture supernatant. Methods The discarded thymus from 18 patients with congenital heart disease undergoing surgical treatment in Department of Cardiothoracic Surgery of Children's Hospital Affiliated to Chongqing Medical University from May 2023 to January 2024 were collected and prepared into thymic slices. After the slices were cultured in vitro for 14 d, scRNA-seq was employed to identify the residual cell types, and gene ontology(GO) and Kyoto encyclopedia of genes and genomes(KEGG) enrichment analysis was performed to analyze the biological function of the residual cells. Then based on the literature concerning thymic slice culture, the molecular markers indicating thymocyte function were screened out. ELISA was applied to detect the changes in protein levels of molecular markers in the supernatant. Receiver operating characteristic(ROC)curve was plotted and assess the value of the molecular markers in the supernatant in evaluating the quality of thymic slices with area under the curve(AUC). Then, the qualified and unqualified thymic slices determined by our obtained molecular markers were transplanted subcutaneously into male nude mice(6~8 weeks old,weighing 14~17 g), respectively, and the male nude mice without transplantation of the thymic slices served as control group. Flow cytometry and histologic analysis were utilized to observe the immune reconstitution after transplantation. Results(1) scRNA-seq identified 11 cell types in thymic slices, dominated with epithelial cells, fibroblasts, and T cells. GO and KEGG enrichment analysis showed that epithelial cells were involved in enrichment entries related to chemotaxis, epithelial cell development, cell matrix adhesion and tight junction;fibroblasts were involved in enrichme

关 键 词：人胸腺组织切片 残余细胞类型 分子标志物 质量评估 

分 类 号：R322.53[医药卫生—人体解剖和组织胚胎学] R361.2[医药卫生—基础医学] R394.3