自发性狼疮肾炎模型小鼠肾小球系膜细胞的原代培养和鉴定  

Primary culture and identification of glomerular mesangial cells from mice with spontaneous lupus nephritis

作　　者：陈鹤鸣 林婷[1] 宋艳芳[1] 林青[1] CHEN Heming;LIN Ting;SONG Yanfang;LIN Qing(People's Hospital Affiliated to Fujian University of Traditional Chinese Medicine,Fuzhou 350004,China)

机构地区：[1]福建中医药大学附属人民医院检验科,福建福州350004

出　　处：《细胞与分子免疫学杂志》2022年第7期611-616,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：福建省自然科学基金(2017J01309)。

摘　　要：目的 原代培养及鉴定自发性狼疮肾炎模型小鼠肾小球系膜细胞,探索分离、培养的最佳条件。方法 对照组ICR小鼠和模型组MRL/lpr自发性狼疮肾炎小鼠各9只,在12、 14和18周龄时,ELISA检测小鼠的自身抗体,二辛可宁酸(BCA)法检测尿蛋白;HE染色和Masson染色观察小鼠狼疮肾炎病变情况。采用细胞筛网分离出肾小球,结合优生选择法原代培养肾小球系膜细胞。通过细胞形态学、免疫细胞化学染色检测α平滑肌肌动蛋白(α-SMA)、足细胞裂隙素(nephrin)表达鉴定MRL/lpr小鼠肾小球系膜细胞。结果 模型组小鼠抗核抗体(ANA)和抗双链DNA(dsDNA)抗体明显增高,尿蛋白在14和18周龄明显增高;MRL/lpr小鼠从12周开始,肾组织即表现出狼疮肾炎相关的病理改变。接种肾小球系膜细胞25 d后,MRL/lpr小鼠肾小球系膜细胞逐渐铺满培养瓶底,相差显微镜下呈星形、梭形。免疫细胞化学显示α-SMA阳性、 nephrin阴性,排除其他肾小球固有细胞。结论 建立了自发性狼疮肾炎小鼠肾小球系膜细胞的原代培养方法并进行了鉴定。Objective To perform primary culture of glomerular mesangial cells, identify them from a mouse model of spontaneous lupus nephritis model and explore the optimal conditions for these procedures. Methods Nine control ICR mice and nine model MRL/lpr mice with spontaneous lupus nephritis were tested for autoantibodies using ELISA and urine protein was detected by bicinchoninic acid(BCA) assay at 12, 14 and 18 weeks of age. HE and Masson staining were used to observe the pathological changes of mice with lupus nephritis. Glomeruli were isolated using a cell sieve, while primary culture of glomerular mesangial cells was performed using the eugenic selection method. Glomerular mesangial cells of the MRL/lpr mice were identified based on cell morphology and immunocytochemical staining of α-smooth muscle actin and nephrin. Results In the model group, levels of anti-nuclear antibody and anti-double-stranded DNA antibodies significantly increased. Urine protein levels also increased significantly at 14 and 18 weeks of age. HE and masson staining of kidney tissue revealed pathological changes associated with lupus nephritis in the MRL/lpr mice from 12 weeks of age. Twenty-five days after seeding of the glomerular mesangial cells, those from the MRL/lpr mice gradually covered the bottom of the culture flasks, appearing star-shaped and fusiform by phase contrast microscopy. Immunocytochemistry showed that these cells were α-smooth muscle actin-positive and nephrin-negative, thus ruling out other glomerular intrinsic cells. Conclusion The primary culture of glomerular mesangial cells from mice with spontaneous lupus nephritis is developed and characterized.

关 键 词：MRL/LPR小鼠 狼疮肾炎 系膜细胞 原代培养 鉴定 

分 类 号：R392-33[医药卫生—免疫学] R692.31[医药卫生—基础医学] R332