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目的研究水甘油通道蛋白(aquaglyceroporins)AQP3、AQP7、AQP9基因在油酸钠诱导的脂肪变性肝细胞模型中的表达变化及其意义。方法以常规培养的人肝癌HepG2细胞为对照,采用油酸钠诱导HepG2细胞脂肪变性,建立非酒精性脂肪变性肝细胞模型,利用油红O染色及细胞内甘油三酯含量测定检测肝细胞脂肪变性程度,并分别于0、12、24、48 h采用实时荧光定量PCR与Western blot方法检测水甘油通道蛋白AQP3、AQP7、AQP9基因的表达。结果油红O染色观察及肝细胞内甘油三酯含量测定显示HepG2细胞在油酸钠处理12 h后即开始出现脂肪变性,随着刺激时间延长,脂肪变性程度逐渐加重,肝细胞内甘油三酯含量在48 h组(79.76±0.75)较0 h组明显升高(P<0.05)。模型组中,AQP3mRNA水平12 h时表达开始减低,48 h时(0.39±0.08)表达最低,与对照组相比差异具有统计学意义(P<0.05);AQP7mRNA表达与对照组相比略有升高,差异无统计学意义(P>0.05);而AQP9 mRNA表达水平自12 h(1.59±0.11)即开始增加,与对照组相比差异具有统计学意义(P<0.05)。模型组中,AQP3蛋白水平12 h时表达开始减低,24、48 h组[(0.016±0.002),(0.012±0.001)]与对照组相比差异具有统计学意义(P<0.05);AQP7蛋白表达与对照组相比较差异无统计学意义(P>0.05);AQP9蛋白水平12 h表达开始增高,24、48 h[(0.050±0.002)、(0.079±0.002)]组与对照组相比差异具有统计学意义(P<0.05)。结论肝细胞脂肪变性模型中水甘油通道蛋白AQP3表达下调、AQP9表达上调、AQP7表达无明显差异,提示不同亚型的水甘油通道蛋白可能通过不同的机制参与了肝细胞非酒精性脂肪变性。
Objective To investigate the changes and significance of AQP3, AQP7 and AQP9 genes in aqua-oleosin-induced steatohepatocytes induced by sodium oleate. Methods Human hepatocellular carcinoma HepG2 cells were used as control. Sodium stearate was used to induce steatosis of HepG2 cells. A non-alcoholic steatohepatocyte model was established. The fatty degeneration of hepatocytes was detected by oil red O staining and intracellular triglyceride assay The expression of AQP3, AQP7 and AQP9 in glycerol channel were detected by real-time fluorescence quantitative PCR and Western blot at 0, 12, 24 and 48 h respectively. Results Oily red O staining and determination of intrahepatic triglyceride content showed that the fatty degeneration of HepG2 cells began 12 h after treatment with sodium oleate. With the prolongation of stimulation time, the degree of steatosis gradually increased. The intracellular triglyceride The content of HGF in 48 h group (79.76 ± 0.75) was significantly higher than that in 0 h group (P <0.05). In the model group, the expression of AQP3 mRNA began to decrease at 12 h and was lowest at 48 h (0.39 ± 0.08), with statistical significance (P <0.05); AQP7 mRNA expression was slightly increased compared with the control group (P> 0.05). However, the expression of AQP9 mRNA began to increase from 12 h (1.59 ± 0.11), which was significantly different from the control group (P <0.05). In the model group, the expression of AQP3 protein began to decrease at 12 h, the difference was statistically significant (P <0.05) between the 24 h and 48 h groups [(0.016 ± 0.002), (0.012 ± 0.001)], and the AQP7 protein (P> 0.05). The expression of AQP9 protein began to increase at 12 h and was significantly lower at 24 and 48 h (0.050 ± 0.002 and 0.079 ± 0.002, respectively) compared with the control group Statistically significant (P <0.05). Conclusions The expression of AQP3, AQP9 and AQP7 in hepatocyte steatosis model were down-regulated, suggesting that different subtypes of glycerol channel protein may participate in the non-alcoholic steatosis of hepatocytes through different mechanisms.