猕猴原代肾小球系膜细胞分离提取培养与鉴定

Isolation,culture and identification of glomerular mesangial cells from macaca mulattas

目的探索一种简单方便、重复性好的非人灵长类动物原代肾小球系膜细胞(GMCs)体外提取、培养及鉴定的方法。方法在无菌条件下取出猕猴肾脏,利用两种不同孔径的纱网(介于100目到200目之间)分离出肾小球,设置不同浓度(0.1、0.2 mg/ml)Ⅵ型胶原酶消化肾小球,同时在这两个浓度基础上设置不同消化时间(10、15 min),将消化后的肾小球接种到细胞瓶中,观察不同消化条件下GMCs的生长状况。显微镜下观察GMCs的结构,免疫荧光和Western blot法检测GMCs中α-平滑肌肌动蛋白(α-SMA)、Nephrin蛋白的表达。利用肿瘤坏死因子-α(TNF-α)刺激后观察GMCs生物学特性,CCK-8法和高内涵细胞成像系统法分别检测GMCs活力和增殖情况;Transwell法和细胞划痕实验检测GMCs迁移能力。结果利用0.1 mg/mlⅥ型胶原酶消化10 min搜集到的肾小球贴壁较多,肾小球细胞生长状态较好。原代培养的肾小球3 d贴壁,7 d开始移出不规则形状或星状的细胞,经21~35 d GMCs逐渐铺满瓶底。GMCs中α-SMA蛋白阳性,Nephrin蛋白阴性。10 ng/ml TNF-α体外刺激能显著增强GMCs的增殖与迁移能力。结论利用胶原酶消化法(0.1 mg/mlⅥ型胶原酶消化10 min)成功建立了GMCs的分离及培养的方法体系,为之后更好模拟人类肾脏疾病提供合适的细胞模型。

Objective To explore a simple,convenient and reproducible method for the in vitro extraction,culture and identification of primary glomerular mesangial cells(GMCs)from non-human primate.Methods The kidneys of macaca mulattas were removed under aseptic conditions,and the glomeruli were separated using two different pore sizes(between 100 and 200 mesh)and digested with type VI collagenase at different concentrations(0.1 mg/ml and 0.2 mg/ml),and different digestion times(10 min and 15 min)were set for the two concentrations.The growth of GMCs under different digestion conditions was observed in the bottles.The structure of GMCs was observed under microscope and the expression ofα-smooth muscle actin(α-SMA)and Nephrin in GMCs was detected by immunofluorescence and Western blot.The biological properties of GMCs were observed after stimulation with tumor necrosis factor-α(TNF-α),and the viability and proliferation of GMCs were detected by CCK-8 and High Connotation Cell Imaging System,respectively andthe migration ability of GMCs was detected by Transwell and cell scratch assay.Results The glomeruli collected using 0.1 mg/ml type VI collagenase digestion for 10 min were more apposed and the glomeruli cells were in a better growth state.Glomeruli in primary culture were adherent at 3 d.Irregularly shaped or star-shaped cells began to migrate at 7 d.GMCs gradually spread to the bottom of the bottle after 21~35 d.GMCs were positive forα-SMA protein and negative for Nephrin protein.10 ng/ml TNF-αin vitro stimulation significantly enhanced the proliferation and migration ability of GMCs.Conclusion The method of collagenase digestion(0.1 mg/ml type VI collagenase digestion for 10 min)was successfully established for the isolation and culture of GMCs,which can provide a suitable cell model to better simulate human kidney diseases in the future.