PTTG1基因在乙醇诱导的LO2肝细胞急性损伤中的作用  

Role of PTTG1 gene in ethanol-induced acute injury of LO2 hepatocytes

作　　者：田轩 沙菲菲 王家玉 郭云蔚[1] Tian Xuan;Sha Feifei;Wang Jiayu;Guo Yunwei(Department of Gastroenterology,the Third Affiliated Hospital of Sun Yat-sen University,Guangzhou 510630,China)

机构地区：[1]中山大学附属第三医院消化内科,广州510630

出　　处：《中华肝脏外科手术学电子杂志》2020年第2期196-200,共5页Chinese Journal of Hepatic Surgery(Electronic Edition)

基　　金：2018年广州市民生科技攻关计划项目(2060404)。

摘　　要：目的探讨PTTG1基因在乙醇诱导的LO2肝细胞急性损伤中的作用。方法LO2肝细胞中加入0、100、200、400、800、1600 mmol/L不同浓度的乙醇,0、3、6、12、24、48 h不同刺激时间进行预实验,镜下观察细胞形态摸索乙醇损伤模型适合的参数。使用携带PTTG1 shRNA的荧光慢病毒载体感染LO2正常人肝细胞系,嘌呤霉素筛选PTTG1敲低稳转株组(PTTG1/KD组),同时建立空载病毒对照组(PTTG1/vector组),两组按摸索参数加入400 mmol/L乙醇作用24 h。HE染色观察各组细胞爬片的形态变化及损伤程度。Western blot检测PTTG1、凋亡相关蛋白Cleaved-Caspase3等的表达情况。多组PTTG1表达比较采用单因素方差分析和LSD-t检验,两组比较采用t检验。结果预实验镜下发现,LO2肝细胞在400 mmol/L及24 h出现明显肝细胞急性损伤形态差异,确定为模型实验参数。PTTG1表达量随乙醇浓度升高和时间增加而增加,于400 mmol/L乙醇浓度、24 h达最高(LSD-t=6.90,4.14;P<0.05)。HE染色发现乙醇组中PTTG1/KD组凋亡细胞较PTTG1/vector组增多。Western blot结果显示,PTTG1/KD组PTTG1表达量为0.22±0.11,明显低于PTTG1/vector组的1.09±0.11(t=-11.60,P<0.05);而PTTG1/KD组的Cleaved-Caspase3表达量为0.05±0.01,明显高于PTTG1/vector组的0.03(t=8.20,P<0.05)。结论乙醇能引起LO2肝细胞PTTG1基因表达变化,而该基因可减轻乙醇诱导的肝细胞凋亡,可能在急性酒精性肝损伤中起到抗凋亡的保护作用。Objective To investigate the role of PTTG1 gene in ethanol-induced acute injury of LO2 hepatocytes.Methods LO2 hepatocytes were treated with ethanol solution of different concentrations 0,100,200,400,800,1600 mmol/L for 0,3,6,12,24 and 48 h,respectively.Cell morphology was observed under microscope to explore the optimal parameters for the establishment of ethanol-induced injury models.Normal human LO2 hepatocyte cell line was infected with fluorescent lentivirus vector carrying PTTG1 shRNA.The stable transfected cell lines with PTTG1 knock-down were screened by Purimycin and assigned in the PTTG1/KD group.The cell lines with empty vector were allocated into the PTTG1/vector group.According to the explored parameters,the cell lines were treated with 400 mmol/L ethanol for 24 h.The morphological changes and severity of injury were observed by HE staining.The expression levels of PTTG1 and apoptosis-related protein of cleaved-caspase3 were detected by Western blot.The expression levels of PTTG1 among multiple groups were statistically analyzed by One-way ANOVA and LSD-t test and the comparison between two groups was conducted using t test.Results Under the microscope in preliminary trial,significant morphological changes of acute injury were observed after exposure to 400 mmol/L ethanol for 24 h.Hence,the experimental parameters of the model were determined.The expression of PTTG1 was up-regulated with the increase of ethanol concentration and treatment time,and reached the highest level when using 400 mmol/L ethanol for 24 h(LSD-t=6.90,4.14;P<0.05).HE staining demonstrated that the number of apoptotic cells in PTTG1/KD group was significantly higher than that in PTTG1/vector group.Western blot showed that the expression of PTTG1 in PTTG1/KD group was 0.22±0.11,significantly lower than 1.09±0.11 in PTTG1/vector group(t=-11.60,P<0.05).However,the expression of cleaved-caspase3 in PTTG1/KD group was 0.05±0.01,significantly higher than 0.03 in PTTG1/vector group(t=8.20,P<0.05).Conclusions Ethanol can cause changes

关 键 词：肝损伤 肝疾病 酒精性 乙醇 PTTG1 

分 类 号：R575[医药卫生—消化系统]