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一种自发胰岛素抵抗的3T3-L1脂肪细胞模型构建 被引量:4

Establishment of a Spontaneous Insulin Resistant 3T3-L1 Adipocyte Cell Model
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摘要 本研究旨在突变3T3-L1细胞葡萄糖转运蛋白4(GluT4)的基因序列,造成GluT4功能障碍,从而获得一种稳定、自发胰岛素抵抗的脂肪细胞模型。利用CRISPR/Cas9技术对3T3-L1细胞的GluT4编码区进行定向切割,通过流式细胞术和基因测序筛选稳定突变的细胞株;使用CCK-8法检测所筛选细胞株的增殖活力;对细胞进行成脂诱导分化,油红O染色以判断细胞成脂分化能力;以胰岛素刺激下葡萄糖摄入量确定细胞对胰岛素的抵抗程度;qPCR和Western blot检测胰岛素抵抗细胞成脂分化过程中关键基因的表达。结果表明:第九外显子处13 bp的缺失导致3T3-L1细胞GluT4蛋白功能障碍,这一突变不影响细胞的增殖活力;成脂诱导分化过程中,该细胞株表现出严重且稳定的自发胰岛素抵抗,葡萄糖摄取受阻,与成脂分化和脂质合成相关的基因表达显著或极显著下调(P<0.05或P<0.01)。综上所述,GluT4功能障碍阻碍了脂肪细胞响应胰岛素的葡萄糖摄取,从而造成脂肪细胞产生自发的胰岛素抵抗;本研究从多个角度对该胰岛素抵抗脂肪细胞模型进行检测分析,验证了其稳定性和有效性,为脂肪营养代谢和胰岛素抵抗相关研究提供了新材料。 The aim of the present study was to obtain a stable, spontaneous insulin-resistant adipocyte model by altering the gene sequence of glucose transporter 4(GluT4) which resulted in the dysfunction of glucose transmembrane transport in 3 T3-L1 cell line. In this study, the GluT4 coding region of 3 T3-L1 was directionally cleaved by CRISPR/Cas9 protein, and the stably mutated cell lines were screened by flow cytometry and gene sequencing. The proliferation activities of the selected cell lines were detected by CCK-8 method. Subsequently, the cells were treated with adipogenic differentiation reagent and identified by oil red O staining. The glucose uptake of cells was detected under basal and insulin-stimulated state to determine the insulin resistant level. qPCR and Western blotting were used to detect the expression of essential genes in the process of adipogenic differentiation of insulin-resistant cells and control cells. The results showed that the 13 bp deletion in the 9 th exon resulted in dysfunction of GluT4 protein in 3 T3-L1 cells without affecting cell proliferation. Sever and stable spontaneous insulin resistance and impaired glucose uptake occurred as we expected. Gene transcription associated with adipogenic differentiation and lipid synthesis were significantly downregulated(P<0.05 or P<0.01) compared with controls. In summary, GluT4 dysfunction hinders insulin-stimulated glucose uptake, resulting in a spontaneous insulin resistance in adipocytes. This study verified the stability and effectiveness of the insulin resistance adipocyte model from multiple perspectives, provided a new material for studies related to adipose metabolism and insulin resistance.
作者 张凯艺 朱文娟 谢宁 叶华琼 杨述林 ZHANG Kaiyi;ZHU Wenjuan;XIE Ning;YE Huaqiong;YANG Shulin(Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China;College of Animal Science,Anhui Science and Technology University,Chuzhou 233100,China)
出处 《畜牧兽医学报》 CAS CSCD 北大核心 2019年第7期1475-1485,共11页 ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金 国家自然科学基金(81770832) 转基因生物新品种培育重大专项(2016ZX08010-003) 中国农业科学院科技创新工程(ASTIP-IAS05)
关键词 葡萄糖转运蛋白4 胰岛素抵抗 3T3-L1细胞 成脂分化 glucose transporter 4(GluT4) insulin resistance 3T3-L1 adipogenic differentiation
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