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

作　　者：刘勇[1,2] 李园[2] 陈珂玲[2] 周斌[2] 杨烈[1,2] 晏会[1,2] 周总光[1,2] 

机构地区：[1]四川大学华西医院胃肠外科中心,成都610041 [2]四川大学华西医院消化外科研究室,成都610041

出　　处：《四川大学学报（医学版）》2012年第5期645-650,共6页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(No.30830100)资助

摘　　要：目的设计并构建编码大鼠葡萄糖调节蛋白78(GRP78)的短发夹样RNA(shRNA)慢病毒载体,用以建立稳定沉默GRP78基因的大鼠胰腺腺泡细胞株AR42J。方法设计并合成3条编码大鼠GRP78mRNA的shRNA质粒表达载体,用三质粒包装系统包装成慢病毒,并以相应慢病毒转导大鼠胰腺腺泡细胞株AR42J。经流式细胞分选术(FCAS)筛选出绿色荧光蛋白(GFP)阳性细胞,采用real-time PCR及Western blot方法检测GRP78基因的mRNA和蛋白表达。结果测序证实,成功构建了编码GRP78的shRNA慢病毒载体LVshGRP78-1、LVshGRP78-2和LVshGRP78-3。慢病毒转导AR42J细胞后,经荧光显微镜和FCAS证实转导效率>85%。经real-time PCR验证,与未处理组相比,LVshGRP78-1、LVshGRP78-2和LVshGRP78-3处理组对GRP78mRNA表达的沉默效率分别为69.4%±1.42%、74.7%±1.69%和86.6%±1.73%(P<0.05),含无义序列的阴性质粒LV-Non Target对照组与未处理组相比,GRP78mRNA的表达差异无统计学意义(P>0.05)。Western blot结果显示各组GRP78蛋白表达与mRNA表达趋势一致。结论成功构建了编码GRP78的shRNA慢病毒载体,建立了稳定沉默GRP78基因的胰腺腺泡细胞株AR42J,为探讨GRP78基因在急性胰腺炎发病机制中的作用提供了新的细胞模型。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 LVshGRP78-1, LVshGRP78-2 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 LVshGRP78 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 GRP78 expression in AR42J cells. This study will provide a new cell model for further study of the role of GRP78 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 AR42J 

分 类 号：R576[医药卫生—消化系统]