槲皮素对肉鸡脂肪细胞内分泌因子水平的影响

摘要

本试验旨在探讨槲皮素对爱拔益加(AA)肉鸡脂肪细胞内分泌因子水平的影响.取10日龄AA肉仔鸡腹部脂肪,采用机械破碎和酶消化相结合的方法分离脂肪细胞,并采用油红O染色和细胞中甘油三酯(TG)含量检测的方法鉴定所分离的细胞.将脂肪细胞随机分5组(每组6个重复),空白组为基础培养液,溶剂对照组在基础培养液中添加2％二甲基亚砜,试验组分别添加10、20、40 mg/L槲皮素,于24、48、72 h收集细胞及培养液,利用酶法测定TG含量,放射性免疫法测定肿瘤坏死因子α(TNFα)含量,酶联免疫吸附测定法测定脂联素(ADP)和过氧化物酶体增殖物激活受体γ(PPARγ)蛋白含量,实时定量PCR法测定PPARγ基因mRNA表达.结果表明:1)对所取脂肪组织进行分离提纯后,显微镜下可见悬浮细胞存在.贴壁后细胞内存在可被油红O染成鲜红色的液滴,同时TG检测显示细胞中存在TG,由此鉴定所取细胞为脂肪细胞.2)基础培养液中添加10、20、40μg/mL槲皮素后,与空白组相比,脂肪细胞TG含量显著降低(P＜0.05)；脂肪细胞中TNFα和ADP含量显著增加(P＜0.05),且存在一定的剂量和时间依赖性；脂肪细胞PPARγ基因mRNA表达和蛋白含量受到抑制(P＜0.05),其中对mRNA表达的影响存在时间和剂量依赖性,而蛋白含量的变化规律不明显.综上所述,槲皮素可通过调节脂肪细胞内分泌因子TNFα、ADP和PPARγ的生成水平来降低脂肪细胞TG含量.%This experiment was conducted to investigate the effects of quercetin on endocrinic factor levels in adipocytes of Arbor Acres (AA) broilers. The methods of mechanical disruption and enzymic digestion were performed to isolate adipocytes from abdominal adipose tissue of 10-day-old AA broilers. The adipocytes were identified by oil red O staining and triglyceride (TG) content. The adipocytes were randomly allocated to 5 groups with 6 replicates each. Adipocytes were cultured in a basal medium (control) , and the basal medium with 2‰ DMSO or 10, 20 and 40 mg/L quercetin. The cells and medium were collected after 24, 48, and 72 h of incubation. Enzymatic method was performed to determine TG content in adipocytes; radioactive immune method was performed to determine tumor necrosis factor (TNFα) content; ELISA was performed to determine the protein content of adiponectin ( ADP) and peroxisome proliferator-activated receptor γ (PPARγ); and real-time polymerase chain reaction (RT-PCR) was performed to determine PPARγ gene mR-NA expression. The results showed as follows: 1) there were abundant suspended cells in microscope through isolation of adipose tissue from AA broilers. The cells were identified as intracellular liquid drop stained by bright red and there was TG in cells. 2) Compared with control, TG content in adipocytes of AA broilers was significantly decreased when the basal medium supplemented with 10, 20, 40 μg/mL quercetin (P <0. 05) ; the contents of TNFa and ADP in adipocytes were significantly increased by the supplementation of 10, 20 and 40 μg/mL quercetin in dose-dependent and time-dependent manner; mRNA expression and protein content of PPARγ were significantly inhibited by the supplementation of 10, 20 and 40 μg/mL quercetin in time-dependent manner but not in dose-dependent manner. The results indicate that the supplementation of quercetin can reduce TG content in adipocytes via regulating the contents of TNFα, ADP and PPARγ in adipocytes of AA broilers.