糖尿病肾病大鼠原代肾小管上皮细胞的提取及体外培养  

Extraction and culture in vitro of primary renal tubular epithelial cells from rats with diabetic nephropathy

作　　者：王慧玲 黄国东[1] 陈宇 刘少芳 王龙龙[1,2] 范氏泰和 WANG Huiling;HUANG Guodong;CHEN Yu;LIU Shaofang;WANG Longlong;PHAM Thithaihoa(Department of Kidney Disease,Guangxi International Zhuang Medicine Hospital Affiliated to Guangxi University of Chinese Medicine,Nanning 530201,Guangxi,China;Graduate School,Guangxi University of Chinese Medicine,Nanning 530200,Guangxi,China)

机构地区：[1]广西中医药大学附属国际壮医医院肾病科,广西南宁市530201 [2]广西中医药大学研究生院,广西南宁市530200

出　　处：《广西医学》2024年第4期535-542,共8页Guangxi Medical Journal

基　　金：广西医学高层次骨干人才培养“139”计划项目(桂卫科教发〔2020〕15号);广西岐黄学者培养项目(桂中医药科教发〔2022〕10号);广西高校中青年教师科研基础能力提升项目(2023KY0291);广西中医药大学全国名中医黄汉儒学术思想与临床诊疗传承发展推广中心项目(2022V004);广西名中医传承工作室建设项目(桂中医药科教发〔2021〕6号)。

摘　　要：目的探讨糖尿病肾病(DN)大鼠原代肾小管上皮细胞(RTECs)的提取及体外培养方法。方法取SD雄性大鼠25只,随机分为正常组(n=5)和模型组(n=20)。对模型组大鼠构建DN大鼠模型,不给予正常组任何处理。获取两组大鼠双肾组织,一部分组织用于HE染色及Masson染色以观察肾组织结构,另一部分用于RTECs的提取及培养。RTECs的提取、培养方法:将肾皮质组织在80目筛网上研磨,经100目筛网过滤、Ⅰ型胶原酶消化25 min后,使用含10%胎牛血清、1%胰岛素⁃转铁蛋白⁃硒⁃乙醇胺添加剂和1%青链霉素双抗溶液的DMEM/F-12完全培养基培养细胞。用免疫荧光染色法检测细胞角蛋白18的表达情况以鉴定所获得的细胞。结果(1)建模后模型组大鼠出现多饮、多食、多尿、体重下降、血糖升高等特征,尿蛋白定性呈阳性,肾组织病理检测提示肾小球结构不完整且出现明显缺损,RTECs明显肿胀、变性,肾小管管腔萎缩,肾小管间质纤维化严重。(2)培养72 h后,两组RTECs均已经贴壁并呈岛屿状聚集生长,培养5~8 d后细胞呈多边鹅卵石样。模型组RTECs高表达细胞角蛋白18,阳性率>95%。结论采用在80目筛网上研磨、100目筛网过滤、Ⅰ型胶原酶消化25 min的方法可以得到数量较多、活性良好、纯度较高的DN大鼠模型RTECs,且在形态与贴壁情况方面与正常大鼠RTECs无明显差异。Objective To explore the extraction and culture in vitro methods of primary renal tubular epithelial cells(RTECs)from rats with diabetic nephropathy(DN).Methods A total of 25 SD male rats were obtained,and they were randomly assigned to normal group(n=5)or model group(n=20).The DN rat model was established on rats of the model group,and no treatment was given to the normal group.Bilateral renal tissues were obtain from the two groups,some tissues were used for HE staining and Masson staining to observe renal tissue structure,and other tissues were used for RTECs extraction and culture.Methods for RTECs extraction and culture were as follows:renal cortical tissues were ground on 80⁃mesh sieve,through 100⁃mesh sieve screening and after 25 minutes of typeⅠcollagenase digestion,DMED/F⁃12 complete medium of 10%fetal bovine serum,1%insulin⁃ferritin⁃selenium⁃ethanolamine additive,and 1%green streptomycin double reactor solution was used to culture cells.The immunofluorescence staining method was employed to detect the expression of cytokeratin 18 to validate the obtained cells.Results(1)After modeling,rats of the model group presented as excessive drinking,eating and urination,and as weight loss,elevation of blood glucose,and other characteristics,as well as positive quantitative urine protein,incomplete glomerular structure and significant defect appearance indicated by renal tissues pathological examination,obvious swelling and degeneration of RTECs,lumen atrophy of renal tubule,and as severe renal tubulointerstitial fibrosis.(2)After 72 hours of culture,both RTECs of the two groups were attached to the wall and grew together in an island pattern,and after 5-8 days of culture,cells presented as multilateral pebbles.RTECs in the model group highly expressed cytokeratin 18,and the positive rate was>95%.Conclusion Employing the method of grinding on 80⁃mesh sieve,screening by 100⁃mesh sieve,and digesting 25 minutes by typeⅠcollagenase can obtain DN rats model RTECs with more quantity,favorable activit

关 键 词：糖尿病肾病 肾小管上皮细胞 原代细胞 细胞提取 细胞鉴定 大鼠 

分 类 号：R587.2[医药卫生—内分泌]