广西医学2024，Vol.46Issue(4)：535-542,8.DOI:10.11675/j.issn.0253-4304.2024.04.13
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
摘要
Abstract
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 type Ⅰ collagenase 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 type Ⅰ collagenase can obtain DN rats model RTECs with more quantity,favorable activity,and high purity.There is no significant difference between DN rats model RTECs and normal rats RTECs in morphology and adhesion condition.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Key words
Diabetic nephropathy/Renal tubular epithelial cells/Primary cells/Cell extraction/Cell validation/Rats分类
医药卫生引用本文复制引用
王慧玲,黄国东,陈宇,刘少芳,王龙龙,范氏泰和..糖尿病肾病大鼠原代肾小管上皮细胞的提取及体外培养[J].广西医学,2024,46(4):535-542,8.基金项目
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) （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) （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) （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) （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) （桂中医药科教发[2021]6号）
