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目的 研究原代骨骼肌细胞中瘦素受体长胞内段mRNA表达增加对葡萄糖氧化的影响。方法 RT－PCR扩增瘦素受体长胞内段cDNA，将扩增产物插入真核表达质粒,构建重组体。重组表达质粒经脂质体介导转染原代骨骼肌细胞，转染后48 h加瘦素和D－［U－14C］葡萄糖，收集14CO2，液体闪烁检测其放射性。另经RT－PCR半定量方法检测瘦素受体长胞内段mRNA在骨骼肌细胞的表达。结果 扩增的瘦素受体长胞内段cDNA及其重组体在限制性内切酶酶切后所得各片段大小均与理论值一致。在重组表达质粒转染组、空表达质粒转染组和非转染组，前者特异性条带和β－actin条带积分光密度比值较后两者升高（均P<0.05）；但3组细胞间葡萄糖氧化差异无显著性。结论 成功构建含有瘦素受体长胞内段cDNA的重组真核表达质粒。单纯增加瘦素受体长胞内段mRNA表达不能改善瘦素对骨骼肌细胞的葡萄糖氧化作用。
Objective To study the effect of increased mRNA expression of leptin receptor in primary skeletal muscle cells on glucose oxidation. Methods RT-PCR was used to amplify the cDNA of leptin receptor endosymbionts. The amplified product was inserted into the eukaryotic expression plasmid to construct a recombinant plasmid. The recombinant plasmids were transfected into primary skeletal muscle cells by liposomes. After 48 h of transfection, leptin and D- [U-14C] glucose were added and 14CO2 was collected. The radioactivity was detected by liquid scintillation. The expression of leptin receptor endosomal mRNA in skeletal muscle cells was also detected by semi-quantitative RT-PCR. Results The size of the fragments amplified by restriction endonuclease digestion of cDNA and recombinant recombinants of leptin receptor endoblasts were consistent with the theoretical values. In the recombinant plasmid transfected group, empty plasmid transfected group and non-transfected group, the ratio of the former specific band and β-actin band integral optical density was higher than the latter two (all P <0.05); but 3 There was no significant difference in glucose oxidation between groups. Conclusion Recombinant eukaryotic expression plasmid containing cDNA of leptin receptor endosymbiostoma was successfully constructed. Simply increasing the expression of leptin receptor endocytic mRNA can not improve the glucose oxidation of leptin to skeletal muscle cells.