四川大学学报（医学版）2012，Vol.43Issue(5)：645-650,6.
稳定沉默GRP78基因的胰腺腺泡细胞株AR42J的建立
Establishment of Pancreatic Acinar Cell Line AR42J with Stable Knockdown of GRP78
摘要
Abstract
Objective To design and construct a lentiviral vector containing shRNA against rat glucose-regulated protein 78 gene (GRP78) , and to establish rat pancreatic acinar cell line with stable knockdown of GRP78 expression. Methods Constructed three plasmid expression vectors coding shRNA against GRP78, and converted them into lentiviral particles using three plasmid package systems. Then, AR42J cells were transduced with produced lentiviral particles. The green fluorescent protein (GFP) positive cells were selected by fluorescence-activated cell sorting (FACS) , the GRP78 gene mRNA and protein expression were detected by real-time PCR and Western blot. Results The DNA sequencing showed that the lentiviral vectors containing shRNA against GRP78 gene were constructed correctly. After the transduction, highly efficient transduction (>85%) of lentivirus in AR42J cells was observed by fluorescent microscopy and FACS. Quantitative real-time PCR showed that GRP78 mRNA expression in AR42J cells was suppressed by L VshGRP78 -1, LVshGRP7S-2 and LVshGRP78-3 lentivirus about 69. 4% ±1.42%, 74. 7%±1. 69% and 86. 6% ± 1. 73% as compared with that of the untreated cells (P< 0. 05) , while LV-Non Target had no significant effect on the GRP78 mRNA level (P>0. 05). Western blot showed that the suppressed effect of LVshG.RP78 lentivirus on GRP78 protein was consistent with GRP78 mRNA. Conclusion The results demonstrated that lentiviral vectors containing the shRNA against GRP78 gene were successfully constructed, which could stably knock down the GRP7& expression in AR42J cells. This study will provide a new cell model for further study of the role of GRP7S in the pathogenesis of acute pancreatitis.关键词
葡萄糖调节蛋白78/RNA干扰/慢病毒/急性胰腺炎/AR42JKey words
Glucose-regulated protein 78/RNA interference/Lentivirus/Acute pancreatitis/AR42J引用本文复制引用
刘勇,李园,陈珂玲,周斌,杨烈,晏会,周总光..稳定沉默GRP78基因的胰腺腺泡细胞株AR42J的建立[J].四川大学学报（医学版）,2012,43(5):645-650,6.基金项目
国家自然科学基金(No.30830100)资助 （No.30830100）
