Université Côte d'Azur, Inserm, C3M, Team Cellular and Molecular Pathophysiology of Obesity and Diabetes, Nice, France;
Université Côte d'Azur, Inserm, C3M, Team Cellular and Molecular Pathophysiology of Obesity and Diabetes, Nice, France.
J Vis Exp. 2021 May 4(171). doi: 10.3791/61925.
Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.
脂肪细胞功能的改变导致了代谢性疾病的发病机制，包括 2 型糖尿病和胰岛素抵抗。这凸显了需要更好地了解脂肪细胞功能障碍所涉及的分子机制，以开发针对肥胖相关疾病的新疗法。调节脂肪细胞中的蛋白质和 microRNA 的表达仍然极具挑战性。本文描述了一种使用小干扰 RNA（siRNA）和 microRNA 模拟物（miR mimic）寡核苷酸通过逆转录转染来分化小鼠成纤维细胞为成熟脂肪细胞，并调节成熟脂肪细胞中蛋白质和 microRNA 表达的方案。该逆转录转染方案涉及将转染试剂和寡核苷酸孵育在细胞培养板中形成复合物，然后向其中加入成熟的脂肪细胞。在存在寡核苷酸/转染试剂复合物的情况下，允许脂肪细胞重新附着在贴壁板表面上。可以对转染的 3T3-L1 成熟脂肪细胞进行胰岛素信号转导、葡萄糖摄取、脂肪生成和脂肪分解等功能分析，以研究蛋白质或 microRNA 操作对脂肪细胞功能的影响。
