摘要
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 a recombinant plasmid of human aquaglyceroporin 9（AQP9）,to examine its expression in liver cell L-02 and to detect its effect on cellular models of nonalcoholic fatty liver disease（NAFLD）.Methods Total mRNA was extracted from human hepatic tissues,and human AQP9 gene was amplified by RT-PCR.AQP9 gene was then inserted into pEGFP-N1 to construct recombinant plasmid pEGFP-N1-AQP9,which was transfected into L-02 cells.The transfection was detected by fluorescent microscopy,and the expression of AQP9 gene was detected by RT-PCR and Western blotting.The effect of AQP9 on NAFLD cellular models was examined by oil red O staining and the determination of triglycerides（TG）,free fatty acids（FFAs） and glycerol contents.Results Recombinant plasmid pEGFP-N1-AQP9 was successfully constructed,and AQP9 expression could be detected after L-02 cells were transfected with pEGFP-N1-AQP9.The oil red O staining showed that the intracellular lipid contents were significantly higher in the oleic acid/pEGFP-N1-AQP9 group than in the oleic acid group（P0.01）.The intracellular TG,FFAs and glycerol contents in the oleic acid/pEGFP-N1-AQP9 group were 5.435±0.337,2.016±0.144 and 1.485±0.113 mmol/L,respectively,while those in the oleic acid group were 3.218±0.220,1.538±0.193 and 1.024±0.148 mmol/L,respectively.It indicated that the upregulation of AQP9 could aggravate the degree of steatosis.Conclusion AQP9 abnormal upregulation can lead to or aggravate NAFLD.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2012年第17期1722-1726,共5页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81070318)
重庆市卫生局医学科学技术研究项目(渝卫科教2010-2-100)~~
