小鼠Foxc2质粒载体的构建及功能鉴定

Construction and Function Identification of Eukaryotic Plasmid Carrying Mouse Foxc2 Gene

构建pcDNA3.1-Foxc2真核表达载体,并以脂质体转染3T3-L1前体脂肪细胞,探究Foxc2对脂代谢的影响。取孕后15d小鼠腹部脂肪组织提取总RNA,用PCR扩增Foxc2全长序列并连接至pMD18-T载体,测序正确后克隆至pcDNA3.1(+)真核表达载体,成功构建pcDNA3.1-Foxc2重组质粒载体。重组质粒转染3T3-L1前体脂肪细胞系,以未转染和空载体转染作为对照,通过RT-PCR和Western blotting法检测Foxc2核酸及蛋白表达,同时检测3T3-L1前体脂肪细胞中脂代谢相关基因FAS、ATGL、HSL及PPARγ的表达。结果显示,重组质粒转染组细胞中Foxc2的核酸和蛋白水平均显著高于对照组。同时发现超表达Foxc2显著降低FAS表达,显著升高HSL及PPARγ的表达,但是对ATGL的表达无显著影响,表明Foxc2具有抑制脂肪沉积的功能。

In order to construct eukaryotic plasmid carrying mouse suppressors of Foxc2 gene, 3T3-L1 preadipocytes were transferred by Lipofection 2000 to preliminary study the lipid metabolism of Foxc2. Total RNA was extracted from mouse subcutaneous fat, plasmids of pMD18-T-Foxc2 and pcDNA3.1-Foxc2 was successfully constructed after PCR and TA cloning. Plasmid pcDNA 3.1- Foxc2 was confirmed by restriction digestion and Foxc2 gene sequencing which was consistent with that of NCBI gene bank. The recombinant plasmid pcDNA3.1-Foxc2 was transfected into 3T3-L1 cell line. The non-transfected 3T3-L1 cells and 3T3-L1 cells transfected with empty vector were served as controls. The mRNA and protein of Foxc2 gene were identified by real-time PCR and western blot analysis. Meanwhile, the expressions of FAS, ATGL, HSL andPPAR7 were detected. The result showed that the mRNA and protein of Foxc2 expression in recombinant pcDNA3.1-Foxc2 group were significantly higher than in un-transfected group and empty vector group. In 3T3-L1 preadipocytes, overexpressionFoxc2 significantly reduced the expression of FAS and significantly increased the expressions of HSL andPPARy , but had little effect on ATGL, which indicated thatFoxc2 functioned to inhibit fat deposition. These results provide the basis for further study of Foxc2 functioned in signaling pathway of fat metabolism in mice 3T3-L1 adipocytes.