人脂联素基因在L-02细胞中表达及其功能检测

Expression and Characterization of Human Adiponectin in L-02 Cells

目的:在人正常肝细胞株L-02中表达外源人脂联素蛋白,并观察其生物学活性。方法:采用脂质体法将成功构建的人脂联素真核表达质粒pcDNA3.1/CT-GFP-TOPO-Adiponectin转染L-02细胞;免疫组织化学定位观察脂联素的表达;Western blot检测培养上清及细胞沉淀中脂联素水平;酶联免疫法测定培养基血糖。结果:构建的真核表达质粒能有效转染L-02细胞,并在培养上清及细胞沉淀中检测到脂联素蛋白,该重组蛋白能够降低高糖处理的L-02细胞的血糖水平。结论:重组人脂联素蛋白能在L-02中有效表达,该蛋白定位在细胞质,并且能分泌到胞外,表达产物经体外实验证实具有生物学活性。

Objective： To express exogenous human adiponectin protein in Homo hepatocytes named L-02,and to observe its bioactivity. Methods： The eukaryotic expression vector pcDNA3.1/CT-GFP-TOPO-Adiponectin was transfected human hepatocytes named L-02 with liposome. The expression of adiponectin protein was observed using immuneohistochemistry. The level of human adiponectin protein in the supernatant of cell culture media and precipitation was detected with western blot, and the culture medium glucose was determined by enzyme linked immunosorbent assay. Results： The eukaryotic expression vector could transfected L-02 effectively. The human adiponectin protein could be detected in culture supernatant, and could significantly reduce the level of blood glucose in hyperglycemia mode of L-02 cells. Conclusion： The recombinant adiponectin protein was successfully expressed in L-02 cells,and the adiponectin protein distributed in cell plasma and succeed to secrete. This expression product has a bioactivity in vitro.