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目的构建人水甘油通道蛋白9(aquaglyceroporin9,AQP9)重组质粒,验证其在L-02肝细胞中的表达,并检测其对非酒精性脂肪肝病(nonalcoholic fatty liver disease,NAFLD)细胞模型的作用。方法从人肝脏组织中提取总RNA,RT-PCR获得AQP9基因,克隆到载体pEGFP-N1上,构建重组质粒pEGFP-N1-AQP9。将其转染L-02细胞,通过荧光显微镜观察其转染情况,RT-PCR和Western blot检测AQP9基因在细胞中的表达。通过油红O染色,测定甘油三酯、游离脂肪酸及甘油含量,检测其对L-02细胞脂肪变性模型的作用。结果成功构建pEGFP-N1-AQP9重组质粒,并能在L-02细胞中表达,将其转染L-02细胞脂肪变性模型后,油红O染色可见油酸/pEGFP-N1-AQP9转染组较油酸组细胞内脂质含量明显升高,油酸/pEGFP-N1-AQP9转染组细胞内甘油三酯、游离脂肪酸及甘油含量分别为(5.435±0.337)、(2.016±0.144)、(1.485±0.113)mmol/L,而油酸组分别为(3.218±0.220)、(1.538±0.193)、(1.024±0.148)mmol/L,两者之间差异有统计学意义(P<0.01),说明上调AQP9表达量可使其脂肪变性程度加重。结论 AQP9异常升高能够引起或加重非酒精性脂肪肝病。
Objective To construct recombinant plasmid of aquaglyceroporin 9 (AQP9) and validate its expression in L-02 hepatocytes, and to investigate its effect on non-alcoholic fatty liver disease (NAFLD) cell model. Methods Total RNA was extracted from human liver tissue. The AQP9 gene was obtained by RT-PCR and cloned into vector pEGFP-N1 to construct recombinant plasmid pEGFP-N1-AQP9. The transfected cells were transfected into L-02 cells. The transfected cells were observed by fluorescence microscope. The expression of AQP9 gene was detected by RT-PCR and Western blot. The content of triglyceride, free fatty acid and glycerol were determined by oil red O staining, and the effect on the model of steatosis in L-02 cells was tested. Results The recombinant plasmid pEGFP-N1-AQP9 was successfully constructed and expressed in L-02 cells. After being transfected into the steatosis model of L-02 cells, the oleic acid / pEGFP-N1-AQP9 transfection group The content of intracellular triglyceride, free fatty acid and glycerol in oleic acid / pEGFP-N1-AQP9 transfection group were (5.435 ± 0.337), (2.016 ± 0.144) and 1.485 ± 0.113) mmol / L respectively, while those in oleic acid group were (3.218 ± 0.220), (1.538 ± 0.193) and (1.024 ± 0.148) mmol / L, Description AQP9 upregulation of the amount of its degree of fatty degeneration. Conclusion AQP9 abnormal increase can cause or aggravate non-alcoholic fatty liver disease.