摘要
目的探讨雷帕霉素对前脂肪3T3-L1细胞脂质稳态、分泌功能的影响,阐明雷帕霉素引起高脂血症的可能机制。方法体外培养前脂肪细胞3T3-L1细胞株,分成对照组、雷帕霉素50、100、200 nmol/L组。用油红O染色(定性)及高效液相色谱法(定量)检测3T3-L1细胞内胆固醇水平,酶联免疫吸附实验分析瘦素、脂联素的分泌量,实时荧光定量PCR法及Western blot法检测3T3-L1细胞过氧化物酶体增殖物激活受体γ(PPARγ)mRNA和蛋白的表达。结果油红O染色显示雷帕霉素组脂肪细胞脂滴数量较对照组明显减少;对照组、雷帕霉素50、100、200 nmol/L组细胞内游离胆固醇含量分别为(12.89±0.16)、(9.84±0.45)、(9.39±0.46)和(8.61±0.34)mg/ml,与对照组相比,雷帕霉素能抑制3T3-L1前脂肪细胞内胆固醇的蓄积(P<0.05)。对照组、雷帕霉素50、100、200 nmol/L组瘦素分泌量分别为(19.02±0.52)、﹙16.98±0.01﹚、﹙15.62±0.01﹚和﹙13.84±0.66﹚ng/ml,与对照组相比,雷帕霉素能减少成熟后3T3-L1脂肪细胞瘦素的分泌水平(P<0.05)。雷帕霉素50、100及200 nmol/L组PPARγmRNA表达量分别为对照组的94%、62%和47%,均显著低于对照组(P<0.05);PPARγ蛋白表达量分别为对照组的80%、74%和61%,均显著低于对照组(P<0.05)。雷帕霉素100 nmol/L、PPARγ阻断剂GW9662 10μmol/L、PPARγ增敏剂曲格列酮10μmol/L分别处理细胞96 h,检测PPARγmRNA的表达分别为对照组的(0.60±0.14)、(0.67±0.03)和(1.30±0.14)倍,与对照组相比,差异具有统计学意义(P<0.05);PPARγ蛋白表达与mRNA的表达变化趋势相似,差异具有统计学意义(P<0.05)。雷帕霉素100 nmol/L、PPARγ阻断剂GW9662 10μmol/L、PPARγ增敏剂曲格列酮10μmol/L分别处理细胞96 h,ELISA检测各处理组瘦素表达量分别为(19.02±0.52)、(15.62±0.10)、(14.45±1.01)和(18.07±0.66)ng/ml,与对照组相比,差异具有统计学意义(P<0.05)。结论雷帕霉素通过下调PPARγ的表达减少脂肪细胞内的
Objective To investigate the effects of rapamycin on cholesterol homeostasis and secretory function of 3T3-L1 cells.Methods The in vitro cultured 3T3-L1 cells（preadipocytes） were divided into control group,rapamycin 50 nmol/L group,rapamycin 100 nmol/L group,and rapamycin 200 nmol/L group.Intracellular cholesterol level was measured by oil red O staining and high performance liquid chromatography.The secretion levels of leptin and adiponectin were assayed by enzyme-linked immunosorbent assay.The mRNA and protein expressions of peroxisome proliferator-activated receptor（PPARγ） were assayed by quantitative real-time polymerase chain reaction and Western blot.Results Oil red O staining showed rapamycin down-regulated 3T3-L1 cells differentiation and lipid accumulation.Quantitative measurement of cholesterol with high performance liquid chromatography showed that the concentrations of free cholesterol in rapamycin treatment groups had a significant reduction.The concentrations of free cholesterol in the control group,rapamycin 50 nmol/L group,rapamycin 100 nmol/L group,and rapamycin 200 nmol/L group were（12.89±0.16）,（9.84±0.45）,（9.39±0.46）,and（8.61±0.34） mg/ml,respectively（P〈0.05）,and the concentrations of total cholesterol were（12.91±0.50）,（9.94±0.96）,（10.45±2.51）,and（9.53±0.63） mg/ml,respectively.The leptin concentrations in the control group,rapamycin 50 nmol/L group,rapamycin 100 nmol/L group,and rapamycin 200 nmol/L group were（19.02±0.52）,（16.98±0.11）,（15.62±0.01）,and（13.84±0.66） ng/ml,respectively.The mRNA expressions of PPARγ in the rapamycin 50 nmol/L group,rapamycin 100 nmol/L group,and rapamycin 200 nmol/L group were significantly lower than that in control group（P〈0.05）.The protein expressions of PPARγ in the rapamycin 50 nmol/L group,rapamycin 100 nmol/L group,and rapamycin 200 nmol/L group were 80%,74%,and 61% of that in control group（P〈0.05）.After the cells were treated with rapamycin 100 nmol/L,PPARγ blocking agen
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
2011年第5期560-565,I0004,共7页
Acta Academiae Medicinae Sinicae
