油酸诱导斑马鱼肝脏细胞脂滴形成与降解模型的建立

Establishment of Lipid Droplets Formation and Degradation Models Induced by Oleic Acid in Zebrafish Hepatocyte

为建立斑马鱼肝脏细胞脂滴形成与降解模型,采用不同浓度油酸孵育肝细胞,通过Alarmablue法及流式细胞术检测油酸对肝细胞存活率、凋亡率的影响,确定最佳油酸浓度,随后用该浓度油酸处理肝细胞72 h,72 h后去除培养基中油酸继续培养观察。采用油红染色法确定脂质蓄积情况,BODIPY/HOECHST染色法观察不同大小脂滴蓄积率,实时定量PCR检测脂滴相关蛋白的转录调控。结果显示,100μmol/L油酸为最佳浓度;与0 h相比,处理6 h小脂滴蓄积率达最大值,48 h大脂滴蓄积率达最大值,72 h超大脂滴蓄积率达最大值;相较于0 h,100μmol/L油酸孵育6~72 h,二酰甘油转移酶基因DGAT2 mRNA水平显著升高;去除油酸24h,脂肪细胞分化蛋白ADRP的mRNA水平显著上调;油酸孵育6 h,脂肪积累诱导跨膜蛋白基因FIT1 mRNA表达水平显著上升;油酸孵育6~72 h以及去除油酸24 h CIDE蛋白家族成员CIDEb mRNA表达均显著升高。由研究可知,油酸诱导斑马鱼肝脏细胞建立脂滴形成与降解模型,可较好的模拟鱼类肝脏脂滴蓄积与降解过程,为研究鱼类肝脏中的脂滴蓄积和降解过程中脂滴蛋白的功能提供了模型基础。

In order to establish zebrafish hepatocytes lipid droplets formation and degradation models,this experiment cultured the zebrafish hepatocytes by different concentrations of oleic acid（ OA） to find out the optimal OA concentration for the subsequent study by measuring cell viability and apoptosis with Alarmablue assay and flow cytometry,respectively. Subsequently,the cells were cultured with media added the optimal OA concentration for72 h. At the end of OA incubation,the cells were cultured with media removed OA. During the OA-supplement and-free periods,the lipid accumulation status was detected by oil red staining and TAG quantify. The cells with different sizes of lipid droplets were observed by BODIPY/HOECHST staining. The percentage of cells with supersized lipid droplets reached the maximun at 72 h. Compared with 0 h,the mRNA level of diacylglycerol acyltransferase 2（ DGAT2） was significantly upregulated from 6 h to 72 h after 100 μmol/L OA exposure. The mRNA level of Adipose differentiation-related protein（ ADRP） was significantly upregulated at 24 h after removing OA. The mRNA level of fat storage-inducing transmembrane protein gene（ FIT1） was significantly upregulated at 6 h after OA exposure. During the whole OA treatment and the OA removing period,the mRNA level of CIDEb,member of CIDE protein family,was significant upregulated. In conclusion,these results demonstrated that the zebrafish hepatocytes models of lipid droplets formation and degradation induced by OA could mimic the process of lipid droplets formation and degradation in fish liver. The models have provided a model basis for further studies on lipid droplets protein.