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原代大鼠肾小球微血管内皮细胞的培养与鉴定

马华根¹, 刘海琴², 刘昭德³, 唐元瑜^4,*

¹北京中医药大学中医学院伤寒教研室，北京 102488；²福建中医药大学中西医结合学院，福州 350122；³南京医科大学基础医学院，南京 211116；⁴福建中医药大学中医学院，福州 350122

摘要

本研究旨在建立一种简便高效的原代大鼠肾小球微血管内皮细胞体外培养方法。摘取出生7~10天Sprague-Dawley大鼠的双侧肾脏，分离出肾皮质；经剪碎，连续过200目、300目筛网获得肾小球；IV型胶原酶消化15~20 min后，收集肾微血管球进行接种培养。通过细胞形态学观察、第VIII因子相关抗原免疫细胞化学染色鉴定所培养的细胞。结果显示：肾微血管球呈不规则球形，无包囊，毛细血管袢结构清晰；静置培养3天后，有短梭形细胞从其周围爬出，并逐渐形成细胞集落，呈“岛屿状”分布；4~5天后细胞集落相互融合，6天后铺满皿底，呈典型的单层、铺路石样、镶嵌式排列。第VIII因子相关抗原免疫细胞化学染色检测结果显示，胞质淡染成棕红色，第VIII因子相关抗原表达为阳性，阳性率接近100%。以上结果提示，本研究将连续过筛法和胶原酶消化法结合，成功建立了简便高效的原代大鼠肾小球微血管内皮细胞体外培养方法，从而为开展糖尿病肾病的发生和发展机理研究提供了重要的工具细胞。

关键词： 肾小球; 微血管内皮细胞; 连续过筛法; 胶原酶消化法; 第VIII因子相关抗原; 大鼠

分类号：R392.33;R33

Primary culture and identification of rat glomerular microvascular endothelial cells

MA Hua-Gen¹, LIU Hai-Qin², LIU Zhao-De³, TANG Yuan-Yu^4,*

¹Department of Shang Han, College of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 102488, China；²College of Integrated Traditional Chinese and Western Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China；³Basic Medical College, Nanjing Medical University, Nanjing 211116, China；⁴College of Traditional Chinese Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China

Abstract

The aim of the present study was to establish a simple and efficient method for isolation and culture of primary rat glomerular microvascular endothelial cells in vitro. The bilateral kidneys were taken from 7–10-day old Sprague-Dawley rats, and the renal cortex was separated. Glomeruli were obtained by cutting and continuously passing 200-mesh and 300-mesh sieves. After type IV collagenase digestion for 15–20 min, renal microvascular globules were collected for inoculation and culture. The cultured cells were identified by cell morphology observation and immunocytochemical staining with factor VIII related antigen. The results showed that the renal microvascular globules were irregularly spherical, without cysts, and the capillary loop structure was clear; after 3 days of primary culture, short spindle-shaped cells crawled out around the renal microvascular globules and gradually formed cell colonies, showing an “island-like shape” distribution; 4–5 days later, the cell colonies fused with each other; 6 days later, the cells covered the bottom of the dish, showing a typical monolayer, paving stone-like, mosaic arrangement. The immunocytochemical staining of factor VIII related antigen showed that the cytoplasm was lightly stained brownish red, and factor VIII related antigen-positive rate of cells was nearly 100%. The above results suggested that this study successfully established a method combining continuous screening and collagenase digestion for culture of primary rat glomerular microvascular endothelial cells in vitro. It provides an important tool cell for studying the mechanism of the occurrence and development of diabetic nephropathy.

Key words: glomerulus; microvascular endothelial cells; continuous screening method; collagenase digestion method; factor VIII related antigen; rats

收稿日期：2021-02-24　　录用日期：2021-05-31

通讯作者：唐元瑜　　E-mail: 2422198977@qq.com

引用本文：

马华根, 刘海琴, 刘昭德, 唐元瑜. 原代大鼠肾小球微血管内皮细胞的培养与鉴定[J]. 生理学报 2021; 73 (6): 926-930.

MA Hua-Gen, LIU Hai-Qin, LIU Zhao-De, TANG Yuan-Yu. Primary culture and identification of rat glomerular microvascular endothelial cells. Acta Physiol Sin 2021; 73 (6): 926-930 (in Chinese with English abstract).