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表达分泌的蛋白)蛋白分泌量伴随体外培养显著下降(P<0.05)。联合IL16和L-selectin生成预测因子Pre1评估体外培养1 d的胸腺组织切片质量的价值最高,ROC曲线下面积为0.883。联合CCL19、CCL21、CXCL12、CXCL16生成预测因子Pre2评估体外培养14 d的胸腺组织切片质量的价值最高,ROC曲线下面积为0.948。(3)裸鼠移植实验证实:与对照组相比,质量检测合格的胸腺组织切片能在体内发育成胸腺结构,并有效提升裸鼠外周血中T细胞的比例(P<0.01),而质量检测不合格的胸腺组织切片移植到裸鼠体内无法重建裸鼠的T细胞发育。结论移植前胸腺组织切片残留的组成细胞主要为上皮细胞、成纤维细胞和T细胞;IL16和L-selectin可作为判定胸腺原料质量的潜在指标。CCL19、CCL21、CXCL12和CXCL16可有效评估胸腺组织切片成品的质量。
Objective To observe the migration and differentiation of the neural precursor cells （NPCs） that derived from murine embryonic stem cells （ESCs） when they were transplanted into amyloid β （Aβ）-treated rat hipp...
详细信息
Objective To observe the migration and differentiation of the neural precursor cells （NPCs） that derived from murine embryonic stem cells （ESCs） when they were transplanted into amyloid β （Aβ）-treated rat hippocampus. Methods MESPU35, a murine ESC cell line that express the enhanced green fluorescent protein （EGFP）, was induced differentiation into nestin-positive NPCs by modified serum-free methods. The Aβ plaques and the differentiation of the grafted cells were observed by immunofluorescent staining. Results Comparing 16 weeks with 4 weeks post-transplantation, the migration distance increased about 5 times; the rate of migratory NPCs differentiating into glial fibrillary acidic protein （GFAP）-positive cells kept rising from （30.41 ± 1.45）% to （49.25± 1.23）%, and the rate of NPCs differentiating into neurofilament 200 （NF200） positive cells increased from （16.68±0.95）% to （27.94± 1.21）%. Meanwhile, the GFAP-positive cells targeting to the ipsilateral side of Aβ plaques increased from 60.2% to 81.3 %, while the NF200-positive cells increased from 61.3% to 84.1%. The migration distance had significant positive linear correlations to the neuronal differentiation rate （r = 0.991） and to the astrocytic differentiation rate （r = 0.953）. Conclusion Engrafted NPCs migrate targetedly to the Aβ injection site and differentiate into neurons and astrocytes.
暂无评论