探讨PTTG1基因在乙醇诱导的LO2肝细胞急性损伤中的作用。

LO2肝细胞中加入0、100、200、400、800、1 600 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。

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）。

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。

To investigate the role of PTTG1 gene in ethanol-induced acute injury of LO2 hepatocytes.

LO2 hepatocytes were treated with ethanol solution of different concentrations 0, 100, 200, 400, 800, 1 600 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.

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).

Ethanol can cause changes of expression of PTTG1 gene in LO2 hepatocytes. PTTG1 gene can alleviate the ethanol-induced apoptosis of hepatocytes, which probably plays a role of anti-apoptosis in acute ethanol-induced liver injury.