Gesta S, Lolmède K, Daviaud D, Berlan M, Bouloumié A, Lafontan M, Valet P, Saulnier-Blache J S
I.N.S.E.R.M U317, Institut Louis Bugnard, Université Paul Sabatier, CHU Rangueil, Bâtiment L3, Toulouse cedex 04, France.
Horm Metab Res. 2003 Mar;35(3):158-63. doi: 10.1055/s-2003-39070.
Primary culture of adipose tissue has often been used to investigate pharmacological and nutritional regulation of adipocyte gene expression. Possible alteration of adipocyte gene expression by primary culture on its own has not been explored in detail. In order to address this issue, explants were prepared from human subcutaneous adipose tissue recovered from plastic surgery and maintained for 0 to 48 h in DMEM supplemented with 10 % serum. At different time points, adipocytes were isolated from the explants by collagenase digestion, and mRNA expression and lipolysis were studied. Culture was associated with an accumulation of tumor necrosis factor-alpha (TNFalpha) in the culture medium, an increase in anaerobic glycolysis, and an increase in the basal lipolysis. In parallel, a rapid and dramatic decrease in the level of mRNA encoding for several adipocyte-specific proteins such as adipocyte lipid-binding protein, hormone-sensitive lipase, lipoprotein lipase, and peroxisome proliferation activating receptor-gamma2 was observed in isolated adipocytes. These downregulations were reminiscent of a dedifferentiation process. In parallel, primary culture was associated with an increase in adipocyte beta-actin, TNFalpha, glucose transporter-1 and hypoxia-induced factor-1alpha mRNAs. Treatment of explants with agents that increase cAMP (isobutylmethylxanthine and forskolin) prevented TNFalpha production and expression and culture-induced alterations of adipocyte gene expression. These data show that primary culture of human adipose tissue explants dramatically alters adipocyte gene expression.
脂肪组织的原代培养常用于研究脂肪细胞基因表达的药理学和营养调控。原代培养自身可能对脂肪细胞基因表达产生的改变尚未得到详细探究。为了解决这个问题，从整形手术中获取的人皮下脂肪组织制备外植体，并在补充有10%血清的DMEM中培养0至48小时。在不同时间点，通过胶原酶消化从外植体中分离出脂肪细胞，并研究mRNA表达和脂解作用。培养与培养基中肿瘤坏死因子-α（TNFα）的积累、无氧糖酵解的增加以及基础脂解作用的增强有关。同时，在分离出的脂肪细胞中观察到几种脂肪细胞特异性蛋白（如脂肪细胞脂质结合蛋白、激素敏感性脂肪酶、脂蛋白脂肪酶和过氧化物酶体增殖激活受体-γ2）编码的mRNA水平迅速且显著下降。这些下调让人联想到去分化过程。同时，原代培养与脂肪细胞β-肌动蛋白、TNFα、葡萄糖转运蛋白-１和缺氧诱导因子-1α mRNA的增加有关。用增加cAMP的试剂（异丁基甲基黄嘌呤和福斯可林）处理外植体可防止TNFα的产生和表达以及培养诱导的脂肪细胞基因表达改变。这些数据表明，人脂肪组织外植体的原代培养会显著改变脂肪细胞基因表达。
