南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (2): 213-218.doi: 10.12122/j.issn.1673-4254.2023.02.08

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一种操作简便的小鼠原代白色成熟脂肪细胞培养方法

申屠志博，弓 欣，杨慧娣   

  1. 内蒙古医科大学基础医学院，研究生院，呼和浩特 内蒙古自治区 010100
  • 出版日期:2023-02-20 发布日期:2023-03-16

A convenient and time-saving method for primary culture of mature white adipocytes from mice

SHENTU Zhibo, GONG Xin, YANG Huidi   

  1. Basic Medical School, Graduate School, Inner Mongolia Medical University, Hohhot 010100, China
  • Online:2023-02-20 Published:2023-03-16

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摘要： 目的 建立一种适合短期培养、操作简便、成本低廉的白色成熟脂肪原代细胞培养的方法。方法 本研究通过分离小鼠附睾处和肾周白色成熟脂肪细胞后，分别采用短期成熟脂肪细胞培养方法和天花板培养方法，培养原代白色成熟脂肪细胞。油红O染色鉴定并观察成熟脂肪细胞细胞形态，CCK8法测定细胞活性，并通过Western blot 检测PPARγ蛋白相对表达量，qPCR检测CD36、FAS、CPT1A、FABP4 mRNA表达量。结果 油红O染色显示改良培养方法细胞形态良好、均一，而天花板法细胞形态出现变化。CCK8显示新鲜分离的白色成熟脂肪细胞与此培养方式细胞活性差异无统计学意义（P=0.959，P=0.657，P=0.694）。Western blot显示新鲜分离的白色成熟脂肪细胞与培养72 h细胞的PPARγ蛋白表达量差异无统计学意义（P=0.759），在使用GW9662处理后显著下降（P<0.001）。qPCR显示，经过GW9662处理后CD36、CPT1A mRNA表达量上调（P<0.001，P=0.003），FAS、FABP4 mRNA表达量下调（P=0.001，P<0.001）。结论 本研究建立了一种培养时间短、操作简单的原代白色成熟脂肪细胞的培养方法，为成熟脂肪细胞的相关研究提供技术保障。

关键词: 原代成熟脂肪细胞；短期培养方法；小鼠

Abstract: Objective To establish a simple, low-cost and time-saving method for primary culture of mature white adipocytes from mice. Methods Mature white adipocytes were isolated from the epididymis and perirenal area of mice for primary culture using a modified mature adipocyte culture method or the ceiling culture method. The morphology of the cultured mature adipocytes was observed using Oil Red O staining, and the cell viability was assessed with CCK8 method. The expression of PPARγ protein in the cells was detected with Western blotting, and the mRNA expressions of CD36, FAS, CPT1A and FABP4 were detected using RT-qPCR. Results Oil Red O staining showed a good and uniform morphology of the adipocytes in primary culture using the modified culture method, while the cells cultured using the ceiling culture method exhibited obvious morphological changes. CCK8 assay showed no significant difference in cell viability between freshly isolated mature white adipocytes and the cells obtained with the modified culture method. Western blotting showed that the freshly isolated adipocytes and the cells cultured for 72 h did not differ significantly in the expression levels of PPARγ protein (P=0.759), which was significantly lowered in response to treatment with GW9662 (P<0.001). GW9662 treatment of the cells up-regulated mRNA expressions of CD36 (P<0.001) and CPT1A (P=0.003) and down-regulated those of FAS (P=0.001) and FABP4 (P<0.001). Conclusion We established a convenient and time-saving method for primary culture mature white adipocytes from mice to facilitate further functional studies of mature adipocytes.

Key words: Primary mature adipocytes; cultural method; mice