抵抗素对3T3-L1脂肪细胞葡萄糖摄取的影响

 【目的】构建载有小鼠抵抗素（resistin）基因及其反义核酸的重组真核表达质粒，探讨抵抗素对3T3-L1脂肪细胞葡萄糖摄取的影响。【方法】用RT-PCR技术从小鼠脂肪组织扩增抵抗素基因cDNA，A／T克隆于pGEM-T载体，再亚克隆于pcDNA3．1（＋）和pcDNA3．1（-）真核表达载体，构建载有小鼠抵抗素（resistin）基因及其反义核酸的重组真核表达质粒pcDNA3．1^（＋）resistin和pcDNA3．1^（-）-resistin^（-）。将pcDNA3．1^（-）resistin^（-）转染小鼠3T3-L1脂肪细胞，通过G418筛选稳定表达的细胞株。3T3-L1脂肪细胞分为对照、瞬时、载体和稳定4个细胞组（每组n=6），用^3H-2-脱氧葡萄糖进行非胰岛素和胰岛素介导的葡萄糖摄取试验。【结果】插入pcDNA3．1^（＋）的DNA片段的核苷酸序列与小鼠抵抗素基因mRNA编码区序列完全一致．且方向正确：插入pcDNA3．10^（＋）的DNA片段的核苷酸序列与抵抗素基因mRNA编码区核苷酸序列完全一致，且方向相反。对照组、瞬时组、载体组和稳定组的3T3-L1脂肪细胞非胰岛素和胰岛素介导的葡萄糖摄取率的无显著性差别（P〉O．05）。【结论】成功构建了载有抵抗素基因和载有抵抗素基因反义核酸的重组真核表达质粒。抵抗素对3T3-L1脂肪细胞的葡萄糖摄取均无明显影响。

Effects of Resistin on Glucose Uptake in 3T3-L1 Adipocytes

Objective To construct respectively the eukaryotic expression plasmids containing a sense and an antisense resistin gene,and study the effects of resistin on glucose uptake in 3T3-L1 adipocytes.Methods Synthesis of resistin cDNA was performed using RT-PCR.The purified PCR product was ligated to pGEM-T vector by T4 DNA ligase.Resistin cDNA subcloned into pcDNA3.1(+) or pcDNA3.1(-) eukaryotic expression vector.The recombination expression plasmids were analyzed nucleotide sequences.The recombinant plasmids pcDNA3.1(-)-resistin(-) were transfected into 3T3-L1 adipocytes;the stable transfection cell line was screened by the culture medium containing G418.3T3-L1 adipocytes were divided into control,transient,vector,and stable groups(n=6),and were experimented glucose uptake with 3H labeled glucose.Results DNA fragment nucleotide sequence inserted pcDNA3.1(+) was consistent with the resistin gene mRNA coding sequence.DNA fragment nucleotide sequence inserted pcDNA3.1(-) was consistent with the resistin gene antisense mRNA coding sequence.There were no significant difference of glucose uptake in both basic state and insulin stimulated among four cell groups(P>0.05).Conclusion The sense and antisenses resistin gene expression plasmids have been successfully cloned.Resistin does not affect glucose uptake in 3T3-L1 adipocytes.