PAX6基因过表达促进大鼠骨髓内皮祖细胞向角膜内皮细胞分化  

Experimental Study of PAX6 Gene Overexpression in Promoting Differentiation of Rat Bone Marrow Endothelial Progenitor Cells into Corneal Endothelial Cells

作　　者：周梦 余菲[1] 邵春益[1] 傅瑶[1] Cells ZHOU Meng;YU Fei;SHAO Chunyi;FU Yao(Department of Ophthalmology,Shanghai Ninth People's Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology,Shanghai 200011)

机构地区：[1]上海交通大学医学院附属第九人民医院眼科,上海市眼眶病眼肿瘤重点实验室,上海市200011

出　　处：《组织工程与重建外科》2021年第1期25-29,共5页Journal of Tissue Engineering and Reconstructive Surgery

基　　金：国家自然科学基金(81570812)。

摘　　要：目的通过研究大鼠骨髓内皮祖细胞转染PAX6基因后角膜内皮细胞相关特征性基因的表达,探讨其作为角膜内皮细胞移植的种子细胞的可能性。方法采用密度梯度离心法分离出大鼠骨髓单核细胞,接种到纤连蛋白铺层的细胞皿,以EGM-2培养液选择培养,获得大鼠骨髓内皮祖细胞。通过免疫荧光染色检测CD31、CD34、CD133、LDL、UEA-1等骨髓内皮祖细胞基因表达情况;病毒转染PAX6基因进入内皮祖细胞,蛋白免疫印迹鉴定转染成功与否;实时荧光定量PCR检测AQP-1、ZO-1、ATP1A1、SNAI1、SNAI2角膜内皮细胞相关特征性基因的表达情况。结果分离获得的细胞在选择培养基中形成类铺路石样克隆,细胞间连接紧密,荧光染色显示内皮祖细胞基因表达且PAX6基因可被成功转染,实时荧光定量PCR检测显示转染后的细胞角膜内皮细胞功能性蛋白基因表达上升,纤维化蛋白基因表达下降。结论大鼠骨髓内皮祖细胞通过过表达PAX6基因可被诱导向角膜内皮细胞转分化,转染后的细胞具有作为角膜内皮细胞移植种子细胞的潜力。Objective To explore the possibility of taking the transfected-PAX6-BEPC(Rat bone-derived endothelial progenitor cells)as the transplanting seed cells by examining the expression of corneal endothelial cells'characteristic genes.Methods Rat monocytes were isolated by density gradient centrifugation,BEPC were selectively cultured by planted on fibronectin coated dish and using EGM-2 culture media.Expression of BEPC specific genes CD31,CD34,CD133,LDL,UEA-1 were detected by cellular immunofluorescence assay.PAX6 gene was transfected by adenovirus and western blotting was used to see whether the transfection succeed or not.q-PCR assay was carried out on transfected cells to detect cornea endothlial cells'characteristic genes(AQP-1,ZO-1,ATP1A1)and fibrosis genes(SNAI1,SNAI2).Results Paving stone-like cell clones formed in the selection medium with tight cell connections,fluorescence staining showed expression of endothelial progenitor cells'gene.PAX6 can be successfully transfected into cultured endothelial progenitor cells,real-time fluorescence quantitative PCR showed the increased expression of functional protein genes in corneal endothelial cells after transfection,with decreased fibrosis protein gene expression.Conclusion Rat bone marrow endothelial progenitor cells can be partially induced to transdifferentiate into corneal endothelial cells through overexpression of PAX6 gene.After transfection,the cells have the potential to be seed cells for corneal endothelial cell transplantation.

关 键 词：骨髓内皮祖细胞 角膜内皮细胞 PAX6 转分化 

分 类 号：R318􀆰[医药卫生—生物医学工程]