【摘要】 目的:分离小鼠原代肝细胞并建立体外糖异生研究模型。方法:利用胶原酶两步灌注法分离并提纯小鼠原代肝细胞,彻底清除细胞内糖原,以丙酮酸和乳酸作为糖异生原料,观察胰高血糖素作用下,小鼠原代肝细胞的葡萄糖产出水平、糖代谢关键酶基因表达水平,以及cAMP-PKA信号通路的激活情况,建立小鼠原代肝细胞糖异生研究模型。结果:分离的小鼠原代肝细胞具有典型的肝细胞形态,可见双核,细胞相互接触,排列成肝索样结构。在胰高血糖素作用下,小鼠原代肝细胞糖异生葡萄糖产出量显著增加,小鼠原代肝细胞糖异生关键酶基因表达明显上调,且胰高血糖素能够促进细胞cAMP积累进而诱导PKA激活。结论:小鼠原代肝细胞分离后状态良好,能够在葡萄糖产出水平及细胞内信号通路激活上高效模拟哺乳动物机体糖异生代谢,是一项体外研究糖异生代谢的有效模型。更多还原

【Abstract】 Objective:To isolate mouse primary hepatocytes and establish a model of gluconeogenesis in vitro.Methods:Using collagenase two-step perfusion method to isolate and purify mouse primary hepatocytes, pyruvate and lactic acid were used as raw materials for gluconeogenesis after the complete removal of intracellular glycogen, and observe the glucose production level, mRNA expression level of key glucose metabolism enzyme genes and cAMP-PKA signaling pathway under the action of glucagon. Establish a stable research model of mouse primary hepatocytes gluconeogenesis.Results:The isolated mouse primary hepatocytes had a typical hepatocyte morphology, with double nucleus, and the cells were in contact with each other and arranged in a hepatic cord-like structure. Under the action of glucagon, the gluconeogenesis glucose output of primary mouse hepatocytes was significantly increased, the expression of key gluconeogenesis enzyme genes in primary mouse hepatocytes was significantly up-regulated, and glucagon can promote the accumulation of cell cAMP and induce PKA activation.Conclusion:Mouse primary hepatocytes are vigorous after isolation, and can efficiently simulate mammalian gluconeogenesis at the level of glucose production and the activation of intracellular signaling pathways. It is an effective model for studying gluconeogenesis in vitro.更多还原