摘要
以改进的两步灌流法分离培养的肝细胞为研究对象，使用含油酸钠0，0．10，0．25，0．50，1．00mmol／L的培养基对其诱导处理12h，通过油红0染色和测定细胞内TG（甘油三脂）含量判断肝细胞脂肪蓄积情况，利用CCK-8法检测细胞的活性和测定细胞培养液中ALT、AST的活性判断细胞活性和损伤程度，选取油酸浓度较为适中的组别电镜观察肝细胞超微结构的变化，荧光定量PCR技术检测油酸钠对肝细胞PPARa，SREBP—1c和ChREBP转录水平的影响，结果显示经过消化、纯化获取高纯度、高活性的肝细胞；生化指标及油红O染色结果显示：0．25mm01／L油酸钠在诱导肝细胞12h后对肝细胞损伤较小，肝细胞保持较高活性，且脂滴较明显。电镜观察结果显示：0．25mmol／L组肝细胞线粒体空泡化严重，糖原颗粒较少的现象符合临床脂肪肝病理现象；关键脂氧化基因PPARa的表达水平升高，脂合成基因SREBP-1c、ChREBP表达水平与对照组明显降低。结果表明：油酸钠诱导奶牛肝细胞12h时，0．25mmol／L浓度的油酸钠为最佳建立体外奶牛脂肪肝细胞模型浓度。
To establish a model of dairy cows hepatocyte steatosis and investigate a possible mechanism for sodium oleate-induced steatosis in dairy cows hepatocytes,normal dairy cows hepatocytes were cultured in different inducing media supplemented with different concentrations of sodium oleate for 12 hours. The activity of the cells was detected by CCK-8 method. The activity Of ALT and AST in the cell culture medium and the amount of TG in the cell were detected. The ultrastructural changes of liver cells were observed by electron microscopy. The transcriptional level of the lipid metabolism gene was detected by real-time quantitative PCR. The results showed that the high purity and high activity hepatocytes were obtained by digestion and purification. Oil red O staining showed that 0.25 mmol/L sodium oleate induced hepatocyte injury after 12 h. Hepatocytes remained high activity and lipid droplets were more obvious. The ultrastructure of hepatocytes in the model group showed cell injury and accumulation of lipid droplets in line with clinical fatty liver pathological findings. The expression of PPARα was increased and the expression level of SREBP-1c and ChREBP was significantly decreased in the control group.. In conclusion, sodium oleate at the concentration of 0. 25 mmol/L was the optimal concentration for establishing the model of dairy cows hepatocyte steatosis.
作者
贾哲
张才
吴庭才
爨淑楠
邵琦
陈小燕
林霖
王宏伟
杨自军
JIA Zhe,ZHANG Cai , WU Ting-cai, CUAN Shu-nan, SHAO Qi, CHEN Xiao-yan, LIN Lin, WANG Hong-wei,YANG Zi-jun(Laboratory of Environmental and Animal Products Safety, Henan University of Science and Technology ,Luoyang, Henan 471003,Chin)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2018年第5期1023-1028,共6页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(31402263)
河南科技大学SRTP项目(2015140)
关键词
奶牛肝细胞
油酸钠
脂肪肝
细胞模型
dairy cow hepatocytes
sodium oleate
fatty liver
cell model
