添加胆固醇对非酯化脂肪酸介导的犊牛肝细胞脂质沉积的差异蛋白组成分析

Analysis of differential protein composition of NEFA-mediated lipid deposition in calf liver cell by adding cholesterol

为了研究胆固醇在非酯化脂肪酸(non-esterified fatty acid, NEFA)介导的犊牛肝细胞脂质沉积中的作用,揭示胆固醇对能量负平衡奶牛肝脂代谢的调控机制,采取体外分离培养犊牛肝细胞,添加NEFA的同时添加不同浓度(20、50和100 mmol/L)的胆固醇,模拟能量负平衡状态下的高NEFA环境,构建脂肪沉积模型。依据细胞生化指标的变化特征确定添加胆固醇的最佳浓度及时间,6 h后以最佳浓度胆固醇继续培养至12 h,确定胆固醇最佳处理时间;收集NEFA组(N组)以及最佳浓度和时间培养的胆固醇+NEFA(N+CHOL组)处理组犊牛肝细胞,利用同位素标记相对、绝对定量(iTRAQ)技术和生物信息学分析方法分析组间的差异蛋白。结果显示:通过对不同方法处理的各组细胞的生化指标进行对比,发现胆固醇处理的最佳浓度为50 mmol/L,最佳时间为6 h;组间差异蛋白共66个,其中有41个蛋白表达上调(比率≥1.2,P<0.05),25个蛋白表达下调(比率≤0.83,P<0.05),这些差异蛋白与黄体酮反应、雌二醇反应、生长激素反应和糖酵解过程有关,相关分子功能有胆固醇结合、钙依赖性蛋白结合、钙依赖性磷脂结合、和D-葡萄糖跨膜转运,其富集的主要代谢通路有Rap1信号通路、氨酰基-tRNA生物合成。

In order to investigate the effect of cholesterol on the NEFA(Non-esterified Fatty Acid)mediated lipid deposition in liver cells of calves and reveal the regulatory mechanism of cholesterol in hepatic lipid metabolism in cows with negative energy balance.Liver cells of calves were isolated and cultured in vitro.Cholesterol at different concentrations(20,50 and 100 mmol/L)was added at the same time simulate the construction of a fat depositon model under the high NEFA condition.The optimal concentration and time of cholesterol addition were determined according to the variation of biochemical indexes.Then,cholesterol of different concentrations was added to NEFA for further culture to determine the optimal concentration and time of cholesterol addition.Six hours later,the optimal concentration of cholesterol was added along with NEFA addition,and culturing continued for 12 h to determine the optimal time for cholesterol treatment.Finally,hepatocytes were collected from the NEFA treated group(the N group)of calves and the cholesterol+NEFA group(the N+CHOL group),followed by analyzing the differential proteins between the two groups using the isotope-labeled relative and absolute quantitative(iTRAQ)technique and the bioinformatic approach.The results were as followed:1)The optimal concentration of cholesterol treatment was 50 mmol/L and the optimal time of addition was at 6 h.2.There were 66 differentially expressed proteins between the two groups,among them 41 proteins were up-regulated(ratio≥1.2,P<0.05).25 were down-regulated(ratio≤0.83,P<0.05).These differentially expressed proteins were related to progesterone reaction,estradiol reaction,growth hormone reaction and glycolysis.The related molecular functions included cholesterol binding,calcium dependent protein binding,calcium dependent phospholipid binding,and d-glucose transmembrane transport.The main metabolic pathways were the Rap1 signaling pathway and biosynthesis of aminoacyl-tRNA.