猪肝星状细胞内质网应激模型的建立及对泛素化的影响

Establishment of Endoplasmic Reticulum Stress Model of Porcine Hepatic Stellate Cell and Its Effect on Ubiquitination

试验对衣霉素(tunicamycin,TM)处理猪肝星状细胞的浓度和培养时间进行筛选,以期成功构建内质网应激(endoplasmic reticulum stress,ERS)模型,并在此基础上探究ERS下猪肝星状细胞泛素化的变化。试验采用浓度为0、1、2、5、10和15μg/mL的TM处理猪肝星状细胞,培养2、4、8、16、24和36 h。通过检测细胞抑制率对TM浓度和培养时间进行初筛;通过检测细胞周期及ERS标志基因和蛋白表达对TM浓度和培养时间进行终选,以确定是否成功构建ERS模型,同时,在ERS下检测猪肝星状细胞泛素化相关基因的表达变化。结果表明:由细胞抑制率检测结果初步确定TM浓度5μg/mL、培养8或24 h后有可能出现ERS。5μg/mL TM在培养8或24 h时,ERS标志基因表达均极显著上调(P<0.01)。在培养8 h时,ERS标志蛋白中仅ATF4显著上调(P<0.05),细胞周期中仅G2/M期细胞比例显著下降(P<0.05);在培养24 h时,ERS标志蛋白均显著上调(P<0.05),细胞周期在G1期出现阻滞,并导致S期和G2/M期细胞比例极显著下降(P<0.01)。以上结果说明,5μg/mL TM培养24 h可成功构建细胞ERS模型。在ERS下,细胞泛素化相关基因UBA 2和UBE 2 E的表达均极显著升高(P<0.01)。综上,猪肝星状细胞经5μg/mL TM处理24 h,可成功建立ERS模型,并启动泛素化机制。

This study was conducted to screen the different concentrations and culture time of tunicamycin(TM)in porcine immortalized hepatic stellate cell,in order to model endoplasmic reticulum stress(ERS)successfully and explore the changes of ubiquitination under ERS.Porcine hepatic stellate cell was treated with TM with concentrations of 0,1,2,5,10 and 15μg/mL,respectively,and cultured for 2,4,8,16,24 and 36 h.TM concentration and culture time were initially screened by detection of cell inhibition rate,and finally selected by detection of cell cycle and ERS marker genes and proteins expression,in order to model ERS successfully.In addition,the expression of ubiquitin related genes in porcine liver stellate cells under ERS were detected.The results showed that 5μg/mL TM culture for 8 or 24 h might model ERS via detection of cell inhibition rate.ERS marker gene expressions were all extremely significantly upregulated by 5μg/mL TM,cultured for 8 or 24 h(P<0.01).In terms of ERS marker protein expression,only ATF4 increased significantly(P<0.05),and only G2/M phase of cell proportion in cell cycle significantly decreased at 8 h of culture(P<0.05).At 24 h of culture,ERS marker proteins were significantly increased(P<0.05),and cell cycle arrest occurred in G1 phase,leading to extremely significant declines in cell proportion in S phase and G2/M phase(P<0.01).The result indicated that ERS model was successfully constructed treated by 5μg/mL TM for 24 h.Ubiquitin related genes UBA 2 and UBE 2 E expressions were extremely significantly increased(P<0.01).In conclusion,the ERS model was successfully established and ubiquitylation mechanism was initiated by cultured with 5μg/mL TM for 24 h in porcine hepatic stellate cell.