CAJ | 학술논문

目的建立稳定表达增强型GFP (EGFP)-LC3的AR42J细胞.方法利用慢病毒过表达载体Lentiviral-EGFP-LC3包装成慢病毒,感染大鼠胰腺腺泡细胞AR42J.以Lentiviral-EGFP感染作为阴性对照.通过倒置荧光显微镜观察及流式细胞分析仪检测病毒感染效率,蛋白质印迹法检测稳定传代细胞株的LC3蛋白表达.用毒胡萝卜素处理细胞建立内质网应激模型,采用蛋白质印迹法检测应激的AR42J细胞的LC3、PERK蛋白表达.结果 AR42J细胞的Lentiviral-EGFP-LC3感染效率＞85％,并能稳定传代.Lentiviral-EGFP-LC3感染的AR42J细胞的LC3 mRNA表达量为未感染细胞的(9.14±0.32)倍;LC3蛋白阳性表达并有EGFP-LC3融合蛋白的表达.Lentiviral-EGFP感染的AR42J细胞的LC3 mRNA表达量为未感染细胞的(1.08 ±0.07)倍,无LC3蛋白表达,仅有GFP表达.与未感染组比较,EGFP-LC3组的LC3 mRNA表达显著升高,差异有统计学意义(P＜0.05),而EGFP组的差异无统计学意义.结论成功构建了稳定表达EGFP-LC3的大鼠胰腺外分泌细胞AR42J,为进一步研究急性胰腺炎与自噬的关系提供了新的细胞模型.
목적 건립은정표체증강형GFP (EGFP)-LC3적AR42J세포.방법 이용만병독과표체재체Lentiviral-EGFP-LC3포장성만병독,감염대서이선선포세포AR42J.이Lentiviral-EGFP감염작위음성대조.통과도치형광현미경관찰급류식세포분석의검측병독감염효솔,단백질인적법검측은정전대세포주적LC3단백표체.용독호라복소처리세포건립내질망응격모형,채용단백질인적법검측응격적AR42J세포적LC3、PERK단백표체.결과 AR42J세포적Lentiviral-EGFP-LC3감염효솔＞85％,병능은정전대.Lentiviral-EGFP-LC3감염적AR42J세포적LC3 mRNA표체량위미감염세포적(9.14±0.32)배;LC3단백양성표체병유EGFP-LC3융합단백적표체.Lentiviral-EGFP감염적AR42J세포적LC3 mRNA표체량위미감염세포적(1.08 ±0.07)배,무LC3단백표체,부유GFP표체.여미감염조비교,EGFP-LC3조적LC3 mRNA표체현저승고,차이유통계학의의(P＜0.05),이EGFP조적차이무통계학의의.결론 성공구건료은정표체EGFP-LC3적대서이선외분비세포AR42J,위진일보연구급성이선염여자서적관계제공료신적세포모형.
Objective To establish a stable AR42J cell line expressing EGFP LC3.Methods The EGFP LC3 overexpressed Lentivirus was constructed and transfected into pancreatic acinar cells (AR42J) of rats.The rats with Lentiviral EGFP transfection were treated as negative control.The transfection efficiency was detected by inverted fluorescence microscope and flow cytometry.The EGFP LC3 protein expression in the stable cell lines were analyzed by Western blot.The cells were treated with thapsigargin to establish endoplasmic reticulum stress model,and the LC3,PERK protein expressions were detected by Western blot.Results The transfection efficiency of Lentiviral EGFP LC3 of AR42J cell was ＞ 85％,which could achieve stable passage.The expression of LC3 mRNA of AR42J cells transfected with Lentiviral EGFP LC3 was 9.14 ±0.32 folds higher than that of negative control,which had no expression of LC3 protein,only EGFP expression.However,compared with non-transfection group,the LC3 mRNA expression in EGFP group was not significantly different.Conclusions A pancreatic acinar cell line (AR42J) of rat stably expressing EGFP LC3 protein is successfully constructed.And it may provide a new model for further research of the relationship between acute pancreatitis and autophagy.