摘要目的 研究胰淀素对脂肪变性人L-02肝细胞甘油三酯代谢的影响及其可能机制.方法 以人L-02肝细胞为实验对象,用含50％胎牛血清的高糖改良杜氏伊格尔培养基(DMEM)孵育肝细胞72 h,制备肝细胞脂肪变性模型.实验分为正常肝细胞组(A组)、脂肪变性肝细胞组(B组)、不同浓度胰淀素孵育脂肪变性肝细胞组:0.01 μmol/L(C组)、0.1μmol/L(D组),1μmol/L(E组)和5μmol/L(F组),胰淀素孵育时间为24 h.油红O染色观察每组肝细胞的形态和肝细胞内脂滴情况,甘油三酯定量检测试剂盒检测每组肝细胞内甘油三酯的含量,逆转录聚合酶链反应(RT-PCR)检测每组肝细胞内脂肪酸合成酶(FAS) mRNA和乙酰辅酶A羧化酶(ACC) mRNA的表达情况.进行正态性和方差齐性检验后,两组间比较采用t检验,多组间比较采用单因素方差分析.结果 用含50％胎牛血清的高糖DMEM培养基孵育肝细胞72 h后,肝细胞胞浆内充满大量红色脂滴,细胞内甘油三酯含量显著升高,说明肝细胞发生脂肪变性.A组甘油三酯含量为(415±52) mg/g蛋白,B组为(1129±96) mg/g蛋白(t=874,P＜0.05)；C、D、E、F组肝细胞内红色脂滴数量减少,甘油三酯含量分别为(898±77)、(740±83)、(515 ±30)、(628±48) mg/g蛋白(F =48.0,P＜0.05).RT-PCR结果显示肝细胞脂肪变性后细胞内FAS mRNA和ACC mRNA表达增加,分别是A组的(1.35 ±0.03)、(1.51 ±0.04)倍,差异具有统计学意义(t=47.2、62.9,P ＜0.05).不同浓度胰淀素处理后各组肝细胞内FAS mRNA和ACC mRNA表达有不同程度的下降.C、D、E、F组FAS mRNA的表达量分别是B组的(0.89 ±0.02)、(0.61 ±0.06)、(0.53 ±0.04)和(1.27±0.01)倍,与B组相比差异均具有统计学意义(t =16.2、58.7、70.4、41.2,均P＜0.05).当胰淀素浓度为5μmol/L时,FASmRNA表达水平较B组升高.C、D、E、F组ACC mRNA的表达量分别是B组的(0.74±0.02)、(0.66 ±0.01)、(0.49 ±0.02)和(1.46±0.05)倍,与B组相比差异均具有统计学意义(t=30.6、40.6、61.1、57.3,均P＜0.05).当胰淀素浓度为5μmol/L时,ACC mRNA的表达水平较B组升高.结论 胰淀素孵育脂肪变性肝细胞后肝细胞内甘油三酯含量下降,脂肪变性程度减轻,可能与肝细胞内FASmRNA和ACC mRNA表达水平变化有关.

abstractsObjective To investigate the effects of amylin on triglyceride metabolism in steatosis human L-02 hepatocyte.Methods The normal hepatoctyes were incubated with 50％ fetal bovine serum high-glucose Ducheune modified Eagle's medium (DMEM) for 72 h to establish the hepatic steatosis model.The experiments were divided into normal hepatocyte cells (Group A),steatosis hepatocyte cells (Group B),steatosis hepatocyte cells incubated with different concentrations of amylin for 24 h (0.01 μmol/L amylin (Group C),0.1 μmol/L amylin (Group D),1 μmol/L amylin (Group E) and 5 μmol/L amylin (Group F)).The oil red O staining was used to observe the hepatocyte morphology and intracellular lipid droplets; the hepatocyte triglyceride quantitative detection kit was used to detect the triglyceride levels in each group (mg/g protein); reverse transcription-polymerase chain reaction was used to examine the synthase mRNA and acetyl coenzyme A carboxylase mRNA expression levels in each group.Comparison between the results of the different groups was performed with either one-way analysis of variance (ANOVA) or Student's t test.Results There were lots of red lipid droplets in hepatocyte cells after incubated with 50％ fetal bovine serum in high-glucose DMEM medium for 72 h ; the triglyceride levels in group A was (415 ±52) mg/g protein,and the content was significantly increased in group B with (1129 ± 96) mg/g protein (t =874,P ＜ 0.05).After incubated with different concentrations of amylin for 24 h,the triglyceride levels in group C,D,E and F were separately (898 ± 77),(740 ± 83),(515 ± 30) and (628 ± 48) mg/g protein (F =48.0,P ＜ 0.05).The red lipid droplets were reduced by oil Red O staining.Reverse transcription-polymerase chain reaction showed that the intracellular fatty acid synthase mRNA and acetyl coenzyme A carboxylase mRNA expression levels were increased in steatosis hepatocytes and reduced after steatosis hepatocytes incubated with different concentrations of amylin.Conclusion When incubated with amylin,triglyceride content in steatosis hepatocytes decreases and the steatosis alleviates and it may be associated with change of the expression levels of fatty acid synthase mRNA and acetyl coenzyme A carboxvlase mRNA.