Prolonged efficiency of siRNA-mediated gene silencing in primary cultures of human preadipocytes and adipocytes.
Prolonged efficiency of siRNA-mediated gene silencing in primary cultures of human preadipocytes and adipocytes.
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DOI：
10.1002/oby.20641
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发表时间：
2014-04
期刊：
影响因子：
6.9
通讯作者：
Puri, Vishwajeet
中科院分区：
文献类型：
--
作者：
Lee, Mi-Jeong;Pickering, R. Taylor;Puri, Vishwajeet
原代人前脂肪细胞和分化的脂肪细胞是研究脂肪形成和脂肪功能与人体脂肪生物学关系的重要细胞培养系统。为了使用这些系统进行机制研究，我们研究了siRNA介导的基因敲除的有效性。开发了有效递送siRNA以在从人皮下脂肪组织分离的原代前脂肪细胞和新分化的脂肪细胞中进行基因沉默的方法。采用定量RT-PCR和蛋白质印迹法测定基因和蛋白质的表达水平。使用显微镜观察脂滴形态，并将甘油释放定量为脂解的量度。siRNA介导的原代人前脂肪细胞基因敲低导致了长期的沉默效应，在整个分化过程中抑制基因。在新分化的脂肪细胞中，siRNA介导的基因敲除允许蛋白质保持耗尽至少5天。有可能在siRNA介导的蛋白质耗尽后重新表达蛋白质。重要的是，siRNA转染的人脂肪细胞保持代谢活性，响应β-肾上腺素能刺激以增加脂解。我们的研究描述了在人前脂肪细胞和脂肪细胞的原代培养物中基因沉默的方法及其延长的有效性。
Primary human preadipocytes and differentiated adipocytes in culture are valuable cell culture systems to study adipogenesis and adipose function in relation to human adipose biology. To use these systems for mechanistic studies, we studied siRNA-mediated knockdown of genes for its effectiveness. Methods were developed to effectively deliver siRNA to for gene silencing in primary preadipocytes isolated from human subcutaneous adipose tissue and newly-differentiated adipocytes. Expression level of genes and proteins was measured using quantitative RT-PCR and western blotting. Lipid droplet morphology was observed using microscopy and glycerol release was quantified as a measure of lipolysis. siRNA-mediated knockdown of genes in primary human preadipocytes resulted in prolonged silencing effects, suppressing genes throughout the process of their differentiation. In newly differentiated adipocytes, siRNA-mediated gene knockdown allowed proteins to stay depleted for at least 5 days. It was possible to re-express a protein after its siRNA-mediated depletion. Importantly, siRNA transfected human adipocytes remained metabolically active, responding to β-adrenergic stimulation to increase lipolysis. Our study describes the methods of gene silencing in primary cultures of human preadipocytes and adipocytes and their prolonged effectiveness.
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影响因子：
7.7
作者：
Pettersson AT;Laurencikiene J;Mejhert N;Näslund E;Bouloumié A;Dahlman I;Arner P;Rydén M
通讯作者：
Rydén M
DOI：
10.1152/ajpendo.00228.2012
发表时间：
2012-11-01
影响因子：
5.1
作者：
Lee, Mi-Jeong;Fried, Susan K.
通讯作者：
Fried, Susan K.
影响因子：
6.5
作者：
Fried, Susan K.;Tittelbach, Thomas;Goldberg, Andrew P.
通讯作者：
Goldberg, Andrew P.
DOI：
10.1152/ajpendo.00265.2004
发表时间：
2005-01-01
影响因子：
5.1
作者：
Tchkonia, T;Tchoukalova, YD;Kirkland, JL
通讯作者：
Kirkland, JL
影响因子：
5.5
作者：
Lee, Joo-Young;Takahashi, Nobuyuki;Kawada, Teruo
通讯作者：
Kawada, Teruo