成熟SVFs细胞与Hepa1⁃6细胞共培养模型的建立及对肝细胞脂代谢的影响
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国家自然科学基金面上项目(91957115);南京医科大学科技发展基金?面上项目(2016NJMU004)


Establishment of co⁃culture of mature SVFs with Hepa1⁃6 as a model for studying effects on lipid metabolism of hepatocytes
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    摘要:

    目的:建立小鼠成熟脂肪细胞血管基质部分(stromal vascular fraction,SVF)与小鼠肝细胞(Hepa1-6)共培养模型,研究成熟脂肪细胞对肝细胞脂质堆积以及代谢的影响。方法:从小鼠皮下脂肪分离提取原代SVF细胞,经体外分化成熟后,以Transwell小室建立成熟SVF细胞和Hepa1-6细胞间接共培养48 h体系。应用逆转录-聚合酶链反应法(RT-PCR法)检测SVF细胞PPARγ和PGC-1α等分化相关基因及Hepa1-6细胞CD36、FATP2、GPAT1等脂代谢相关基因的表达。酶法检测经共培养后的Hepa1-6细胞甘油三酯水平。结果:小鼠原代SVF细胞经诱导成熟后,出现明显较大脂滴;成熟SVF细胞中PPARγ和PGC-1α等分化标志物较未分化SVF细胞显著升高;Hepa1-6细胞甘油三酯水平明显高于未共培养组;Hepa1-6细胞GPAT1、CD36基因表达水平明显升高。结论:已成功建立成熟脂肪细胞与Hepa1-6细胞Transwell共培养模型。肝细胞与成熟脂肪细胞共培养可以诱导其脂质堆积增加,该过程可能通过上调肝细胞中CD36 和GPAT1的表达水平增加脂肪酸的吸收和甘油三酯的合成通路实现。

    Abstract:

    Objective:This study aims to establish a co-culture system of matured stromal vascular fraction (SVF) with Hepa1-6 and study the effects on lipid metabolism of hepatocytes. Method:Primary SVF cells were isolated from mice subcutaneous fat and induced to matured SVF cells with differentiation medium. Matured SVF cells were co-cultured with Hepa1-6 by using Transwell system for 2 days. RT-PCR was applied to detect the expression of differentiation biomarker such as PPARγ and PGC-1α in SVF cells and lipid metabolism related genes such as CD36,FATP2 and GPAT1 in Hepa1-6. Cellular triglyceride level of co-cultured Hepa1-6 was detected by GPO Enzyme Method. Results:Isolated primary SVF cells were successfully differentiated to mature SVF cells with significantly large lipid droplets after treated with adipocytes differentiation medium for 6 days. The expression level of PPARγ and PGC-1α of SVF cells was significantly increased. The cellular triglyceride level of co-cultured Hepa1-6 was significantly higher than that of control group accompanied with increased expression level of GPAT1 and CD36. Conclusion:The co-cultured system of matured SVF cells with Hepa1-6 in Transwell insert has been successfully established. Co-culture with mature SVF cells induces lipid accumulation in Hepa1-6. This process may be maintained by the elevated expression level of CD36 and GPAT1,increasing fatty acid absorption and triglyceride synthesis.

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王雨竹,张 许,李 仲.成熟SVFs细胞与Hepa1⁃6细胞共培养模型的建立及对肝细胞脂代谢的影响[J].南京医科大学学报(自然科学版),2022,42(5):603-609

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  • 收稿日期:2022-03-03
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  • 在线发布日期: 2022-05-23
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