小鼠永生化心内膜细胞系的建立与鉴定

Establishment and Characterization of an Immortalized Endocardial Cell Line in Mouse

目的构建小鼠心内膜体外培养模型,促进心内膜在心脏损伤和修复中的机制研究。方法利用酶消化法获得心室游离壁原代心内膜细胞,利用RT-qPCR和免疫荧光对心内膜相关标志物进行鉴定;之后通过SV40-LT-GFP-PURO慢病毒感染分离得到的原代心内膜细胞,经过嘌呤霉素筛选、传代扩增、CD31磁珠分选纯化后,获得永生化的小鼠心内膜细胞系,并对其进行形态学评估、分子标志物检测和功能学验证。结果分离得到的原代心内膜细胞呈现鹅卵石样细胞形态,表达心内膜特异性基因和蛋白,但是数量少且传代次数有限。永生化的小鼠心内膜细胞系表达心内膜特异性基因,保留了体外血管生成和乙酰化低密度脂蛋白摄取的功能特性,且能维持与原代心内膜相似的鹅卵石样形态至少传代15次。结论本研究成功构建了能多次传代且保留心内膜特异性基因表达特征和内皮细胞功能特性的小鼠永生化心内膜细胞系。

Objective To establish an in vitro culture model of mouse endocardium,so as to promote the study of endocardial mechanisms in cardiac injury and repair.Methods Primary endocardial cells were isolated from the inner wall of the ventricular free wall by enzymatic digestion,and the expression of endocardial-specific markers were identified using RT-qPCR and immunofluorescence.SV40-LT-GFP-PURO lentivirus was transduced into the isolated primary endocardial cells,and an immortalized mouse endocardial cell line was obtained after puromycin selection,expansion,and purification by CD31 magnetic beads sorting.Then morphological evaluation,molecular marker detection and functional validation of this immortalized endocardial cell line were processed.Results Primary endocardial cells showed a cobblestone-like cell morphology and expressed endocardium-specific genes and proteins,but in small numbers and with limited capability of passaging.The immortalized mouse endocardial cell line expressed endocardial-specific genes and retained the functional characteristics of angiogenesis and acetylated LDL uptake in vitro.It can be passaged at least 15 times and maintained a cobblestone-like morphology similar to that of the primary endocardium cell.Conclusion In this study,we successfully established a mouse immortalized endocardial cell line that is capable of multiple passages and retains endocardial-specific gene expression and functional properties of endothelial cells.