脂联素对脂肪细胞条件培养基促系膜细胞合成转化生长因子β1的作用

Adiponectin inhibits transforming growth factor β1 upregulation induced by adipocyte conditioned medium in mouse mesangial cells

目的 探讨脂联素对脂肪细胞条件培养基促系膜细胞（MsMC）合成转化生长因子β1（TGFβ1）的作用.方法 将MsMC分为7组：A组加入无血清DMEM培养基;B组加入脂肪细胞条件培养基;C组加入pSuper质粒转染后条件培养基;D组加入pcDNA3.1（-）质粒转染后条件培养基;E组加入脂联素siRNA-pSuper表达质粒转染后条件培养基;F组加入脂联素pcDNA3.1（-）表达质粒转染后条件培养基;C组加入添加3 mg/L脂联素的脂肪细胞条件培养基.用脂联素pcDNA3.1（-）表达质粒、脂联素siRNA-pSuper表达质粒、pcDNA3.1（-）质粒及pSuper质粒分别转染3T3-L1细胞（脂质体2000转染法）,诱导细胞分化为成熟脂肪细胞后收集细胞,以蛋白质印迹法检测细胞裂解液中脂联素蛋白含量.孵育MsMC 9 h后,收取细胞,用实时定量聚合酶链反应检测细胞裂解液中TGFβ1 mRNA水平;孵育24 h后收取上清液,用酶联免疫吸附试验检测上清液中TGFβ1蛋白质水平.结果 与未转染的成熟脂肪细胞比较,用脂联素pcDNA3.1（-）表达质粒转染3T3-L1细胞得到的成熟脂肪细胞的细胞裂解液中脂联素含量增加[（0.59±0.06）比（0.21±0.02）],差异有统计学意义（P＜0.05）;用脂联素siRNA-pSuper表达质粒转染3T3-L1细胞得到的成熟脂肪细胞的细胞裂解液中脂联素含量减少[（0.12±0.01）比（0.39±0.02）],差异有统计学意义（P＜0.05）.B组TGFβ1 mRNA水平及蛋白质表达含量高于A、F、G组[TGFβ1 mRNA：（3.09 ±0.15）比（1.50±0.35）、（2.1±0.53）、（1.8±0.42）,TGFβ1蛋白质水平：（708±10）比（224±16）、（382±11）、（300 ±25）],差异有统计学意义（P＜0.05）.结论 脂肪细胞条件培养基可刺激系膜细胞合成TGFβ1,而脂联素对这一效应具有明显抑制作用.

Objective To investigate if adiponectin has an inhibitive effect on transforming growth factor β1 （TGFβ1） up-regulation in mouse mesangial cells induced by adipocyte conditioned medium.Methods Mouse mesangial cells were divided into the following groups：group A had serumfree DMEM medium; group B had fat cell conditioned medium; group C had fat cell transfected with pSuper gene conditioned medium; group D had fat cell transfected with pcDNA3.1 plasmid conditioned medium; group E had fat cell transfected with adiponectin siRNA-pSuper expression plasmid conditioned medium; group F had fat cell transfected with adiponectin pcDNA3.1 expression plasmid conditioned medium;group G had fat cell conditioned medium including 3 mg/L adiponectin.After the mesangial cells were incubated for 9 h,TGFβ1 mRNA in cell lysate was detected by real time PCR; after incubation for 24 h,TGFβ1 protein in supernatant was measured by ELISA.Results Compared with non transfected mature fat cells,the content of adiponectin in cell lysates from mature fat cells which were transfected by adiponectin pcDNA3.1 （-） expression plasmid was significantly increased[（0.59 ± 0.06） vs （0.21 ± 0.02）] ; the content of adiponectin in cell lysates from mature fat cells which were transfected by adiponectin siRNA-pSuper expression plasmid was significantly decreased [（0.12 ± 0.01） vs （0.39 ± 0.02）] （P ＜ 0.05）.Compared with group B,the mRNA and protein expression of TGFβ1 was significantly down-regulated in the group A,F,G [TGFβ1 mRNA：（3.09 ±0.15） vs （1.50±0.35）,（2.1 ±0.53）,（1.8 ±0.42） ;TGFβ1 protein：（708 ± 10） vs （224 ± 16）,（382±11）,（300 ±25）,P＜0.05].Conclusion Adipocyte CM can significantly up-regulate TGFβ1 expression in mouse mesangial cells and adiponectin can signific antly inhibit this effect.