Resistin concentrations in murine adipose tissue and serum measured by a new enzyme immunoassay
Resistin concentrations in murine adipose tissue and serum measured by a new enzyme immunoassay
复制标题
DOI：
10.1038/oby.2006.26
复制
发表时间：
2006-02-01
期刊：
影响因子：
6.9
通讯作者：
Ohta, Mitsuhiro
中科院分区：
文献类型：
--
作者：
Fujinami, Aya;Ohta, Kiyoe;Ohta, Mitsuhiro
目的：为了澄清抵抗素基因在脂肪组织和血清中的表达与蛋白质分析之间的矛盾，我们建立了一种灵敏的直接检测小鼠抵抗素的酶联免疫吸附试验(EL ISA)。然后，亲和纯化的抗C末端抵抗素免疫球蛋白G(Ig G)被生物素标记。酶联免疫吸附试验(EL ISA)是基于包被在聚苯乙烯平板上的抗体Ig G和生物素标记抗体Ig G之间的抗原夹心。用链霉亲和素标记的辣根过氧化物酶对结合的生物素化抗体进行定量。结果：新的酶联免疫吸附试验可检测到低至0.5 ng/mL的重组小鼠抵抗素，并具有足够的敏感性和特异性，可用于各种脂肪组织和血清中抵抗素蛋白的检测。在正常小鼠中，白色脂肪组织和血清中抵抗素浓度的下降在发育6到24周期间都与年龄有关。与肾周和腹部脂肪组织相比，大网膜脂肪组织中抵抗素浓度显着升高，在生长期内，女性的抵抗素浓度是男性的2-5倍。Ob/ob小鼠的血清和白色脂肪组织中的抵抗素浓度均显著低于对照组，尤其是在大网膜脂肪中。用睾酮而不是孕酮或β-雌二醇处理培养的脂肪细胞，以剂量依赖的方式降低抵抗素蛋白水平。讨论：新的灵敏的小鼠抵抗素酶联免疫吸附试验表明，与肾周和腹部脂肪细胞相比，正常小鼠的大网膜脂肪库中的抵抗素水平显著升高，而与遗传性肥胖小鼠的脂肪组织相比，正常小鼠的抵抗素水平显著升高。
Objective: In an attempt to clarify the conflicting data on resistin mRNA expression and protein analysis by western blotting in adipose tissue and serum, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) for direct measurement of mouse resistin.Research Methods and Procedures: We developed polyclonal antibodies directed to the N (21 to 40) and C (79 to 91) termini of mouse resistin. Then, affinity-purified anti-C-terminal resistin immunoglobin G (IgG) was biotinylated. ELISA was based on the sandwiching of antigen between antibody IgG coated on polystyrene plates and biotinylated antibody IgG. The bound biotinylated antibody was quantified with streptavidin-linked horseradish peroxidase.Results: New ELISA can measure a concentration as low as 0.5 ng/mL of recombinant mouse resistin and is sensitive and specific enough to measure resistin protein in various adipose tissues and in sera. In normal mice, decreases in resistin concentrations in both white adipose tissue and serum were age dependent during 6 to 24 weeks of development. Resistin concentrations were significantly higher in omental adipose tissue in comparison with perirenal and abdominal adipose tissues and were 2- to 5-fold higher in females than males during the growth period. ob/ob mice had significantly lower resistin concentrations than the control mice in both sera and the white adipose tissues, particularly in the omental fat. The treatment by testosterone, but not progesterone or beta-estradiol, in cultured adipocytes reduces resistin protein levels in a dose-dependent manner.Discussion: New sensitive ELISA for mouse resistin clarified that the resistin concentrations in normal mice were markedly elevated in the omental adipose depots as compared with the perirenal and abdominal adipocyte depots and significantly elevated compared with adipose tissues in genetically obese mice.