基于预孵法纯化培养小鼠原代视网膜微血管周细胞  

Purification and cultivation of mouse primary retinal microvascular pericytes based on pre-incubation

作　　者：刘光辉[1] 林翠红 杨田野 徐朝阳[1] 郑永征[1] 赵利[1] 孟春[2] 潘铭东[1] Liu Guanghui;Lin Cuihong;Yang Tianye;Xu Chaoyang;Zheng Yongzheng;Zhao Li;Meng Chun;Pan Mingdong(Affiliated People's Hospital,Fujian University of Traditional Chinese Medicine,Fuzhou 350004,China;College of Biological Science and Biotechnology,Fuzhou University,Fuzhou 350108,China)

机构地区：[1]福建中医药大学附属人民医院眼科,福州350004 [2]福州大学生物科学与工程学院,350108

出　　处：《中华实验眼科杂志》2019年第10期774-778,共5页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目(81774369);福建省自然科学基金项目(2016J01574);福建省医学创新项目(2015-CXB-23).

摘　　要：目的建立简便的小鼠原代视网膜微血管周细胞(RMPs)分离、纯化、培养方法.方法在体视显微镜下机械分离获取小鼠视网膜,并碎化、胶原酶消化、过滤处理,收集视网膜碎片,预孵24h后用接种于含有体积分数20%胎牛血清的低糖型DMEM6孔板进行培养,并采用差异消化法纯化原代RMPs.通过倒置相差显微镜观察细胞形态,免疫荧光法鉴定周细胞标志物,周细胞/内皮细胞共培养系统鉴定周细胞的功能.结果经预孵的视网膜碎片再次孵育24h后,细胞开始从视网膜碎片中迁移出,逐渐增生形成大小不一的细胞集落.原代或子代细胞胞体呈不规则三角形,多个长突触,无接触性抑制.免疫荧光显示第3代细胞绝大部分α平滑肌肌动蛋白(α-SMA)、血小板源性生长因子受体β(PDGFR-β)表达阳性,极少部分胶质纤维酸性蛋白(GFAP)表达阳性,vonWillebrand因子(vWF)表达阴性,RMPs纯度达97%以上.体外培养的小鼠RMPs能被血管内皮细胞所募集,共同形成微血管样条索.结论本实验成功建立了一种简便的小鼠原代RMPs分离、纯化、培养方法,所培养的小鼠原代RMPs为功能性RMPs.Objective To establish a simple method for isolation, purification and cultivation of primary retinal microvascular pericytes (RMPs) from mice. Methods Retinas were isolated from mice following with mechanical morcel, enzymatic digestion and filtration.The retinal fragments were incubated with low glucose DMEM with 20% fetal bovine serum after 24 hours pre-incubation.Differential digestion was used for purification of primary RMPs.Morphological examination of cells was performed by phase contrast microscopy, and further characterization was analyzed by immunocytochemistry.Functional assay was evaluated by the pericytes-endothelial cells (ECs) co-culture system.The treatment and use of experimental animals followed the regulations on the administration of experimental animals promulgated by the state science and technology commission. Results Cells migrated out of fragments after 24 hours of incubation, and developed into small or large colonies gradually.The cells and their subpassages presented typical pericyte morphology with large irregular triangular cell bodies and multiple long processes.No contact inhibition was observed.Most cells uniformly expressed the cellular markers α-smooth muscle actin (α-SMA) and platelet-derived growth factor receptor-β(PDGFR-β), a few cells expressed the cellular markers glial fibrillary acidic protein (GFAP), but no cell expressed von Willebrand factor (vWF). The purity rate of RMPs was up to 97%.In the co-culture system, RMPs directly contacted with ECs to form the capillary-like cords in vitro. Conclusions A simple method for the isolation, purification cultivation of mouse RMPs is established, and active RMPs can be readily obtained by this method.

关 键 词：视网膜微血管周细胞 预孵 分离 纯化 培养 

分 类 号：R774.1[医药卫生—眼科]