【作者】 欧健；

【导师】 杨钦河；

【作者基本信息】 暨南大学 ， 中西医结合临床， 2009， 硕士

【摘要】 目的:观察大黄素对脂肪变性L02肝细胞UCP2 mRNA表达的影响,探讨其对脂变肝细胞的降脂作用和机制。方法:1.复制模型:以L02肝细胞为研究对象,用含50%小牛血清的1640培养基孵育L02肝细胞48h,建立肝细胞脂肪变性模型,用油红O染色,倒置显微镜观察肝细胞的脂滴积聚情况,检测细胞培养上清肝酶(ALT、AST、ALP、LDH)和细胞内甘油三酯(TG)的变化。2.筛选药物浓度:采用MTT法测定大黄素作用于脂肪变性肝细胞的适宜浓度。3.指标检测:模型复制成功后,分为4组:模型组、自然恢复组、大黄素高剂量组、大黄素低剂量组,同时设立正常对照组。药物作用24h、48h后,分别采用全自动生化分析仪检测肝细胞内TG含量的变化。采用RT-PCR法检测各组孵育24h后肝细胞內UCP2 mRNA的表达。结果:1.肝细胞脂肪变性模型:成功建立了高浓度小牛血清诱导的L02肝细胞脂肪变性模型,油红O染色显示,模型组在培养24h、48h、72h后,细胞内有逐渐增多的橘红色和红色的脂滴出现,而正常组细胞内则少见。与正常组比较,造模24h后细胞上清AST、LDH含量显著升高(P＜0.01),ALT、ALP未见有显著变化(P＞0.05),肝细胞中TG含量明显升高(P＜0.01);在48h、72h时段,则ALT、AST、ALP、LDH和TG水平都显著升高(P＜0.5,或P＜0.01)。2.肝脂:大黄素治疗24h后,自然恢复组肝细胞內TG含量与模型组比较明显降低(P＜0.01);与自然恢复组比较,大黄素10μM组肝细胞内TG含量明显降低,差异有统计学意义(P＜0.05),而5μM组无明显降低(P＞0.05)。油红O染色结果显示,模型组细胞浆内见大量橘红色和红色的脂滴,正常组、自然恢复组和大黄素治疗组细胞浆内脂滴数和脂滴的颜色较模型组明显减少,其中大黄素10μM组减少最明显。大黄素治疗48h后,自然恢复组和各治疗组之间无显著性差异(P＞0.05)。油红O染色结果显示,模型组细胞浆内见大量橘红色和红色的脂滴,正常组、自然恢复组和大黄素治疗组细胞浆内脂滴数和脂滴的颜色较模型组明显减少。但大黄素各治疗组与自然恢复组之间比较镜下观察无明显差异。3.肝细胞UCP2 mRNA的表达:大黄素治疗24t3后,与正常组比较,模型组肝细胞UCP2mRNA表达明显上升,差异有显著性(P＜0.01);各治疗组与自然恢复组比较,UCP2 mRNA表达无明显下降(P＞0.05)。结论:1.高浓度小牛血清可诱导肝细胞的脂肪变性:控制高脂因素对肝细胞的脂肪变性程度有改善作用。2.大黄素可降低脂肪变性肝细胞的TG水平。3.脂质在肝细胞内的蓄积可能与细胞内UCP2 mRNA表达上调有关。大黄素降低TG的作用可能与脂质代谢其他相关基因的调控有关。更多还原

【Abstract】 Objective:To observe the effects of Emodin on expression of UCP2 mRNA in steatotic hepatocytes,to explore the possible mechanism in treating nonalcoholic fatty liver disease.Methods:1.Establishing the model of hepatocyte steatosis:A model of hepatocyte steatosis was established by 50%calf serum.Lipid droplets in hepatocytes were observed by inverted microscope after oil red-O staining.The levels ofALT,AST,ALP,LDH in the medium and TG in the hepatocytes were detected.2.Selecting the appropriate concentration of emodin,the appropriate concentration and treatment time were selected by MTT assay.3.Index detecting:After the model of hepatocyte steatosis was established,it was divided into 5 groups:normal group,model group,spontaneous recovery group,emodin group with the concentration of 5μM and 10μM..The level of TG in the hepatocytes was detected by automatic biochemical analyzer afer emodin treating 24h and 48h respectively.The expression of UCP2 mRNA in each group was detected by RT-PCR.afer emodin treating 24h.Results:1.Steatosis model:The model of hepatocyte steatosis was established by 50%calf serum successfully.Orange red and red lipid droplets were observed by inverted microscope in the model group after oil red-o staining,but there were less in the normal group.Compared with the normal group,the levels of AST,LDH and cellular TG were increased in the 24h model group(P＜0.01),but the levels of ALT and ALP presented no significant difference(P＞0.05).The levels of ALT,LDH,AST,ALP and the content of TG in the hepatocytes were increased significantly in the 48h and 72h model group(P＜0.01,P＜0.05).2.Content of TG:After cultured with emodin for 24h,compared with the model group, content of TG in spontaneous recovery group were decreased significantly(P＜0.01), compared with the spontaneous recovery group,content of TG in 10μM emodin group was decreased significantly(P＜0.05),5μM emodin group presented no significant difference (P＞0.05).Orange red and red lipid droplets were observed in the model group;Compared with the model group,orange red and red lipid droplets were less in normal group, spontaneous recovery group,5μM emodin group and10μM emodin group.After cultured with emodin for 48h,compared with the spontaneous group,the treatment groups presented no significant difference(P＞0.05).Orange red and red lipid droplets were observed in the model group,other groups were less than mode! group,but it presented no difference between spontaneous recorvery group and treatment groups observed by inverted microscope.3.The expression of UCP2 mRNA:Compared with the normal group,the expression of UCP2 mRNA in the hepatocytes of model group was higher which was statistically significant(P＜0.01).Compared with the spontaneous recorvety group,expression of UCP2 mRNA in hepatocytes of treatment groups presented no significant difference(P＞0.05).Conclusion:1.High consistence serurn could induce steatosis hepatocyte model;controling high consistence serum was significant in ameliorate steatosis status.2.Emodin could degrade the level of TG in steatotic hepatocytes.3.The deposition of lipid in hepatocytes might be relative with the upregulate of UCP2 mRNA.The function of decreasing cellular TG by emodin might have the participance of other genes correlated with lipid metabolism.更多还原