慢病毒介导的稳定沉默MyD88基因的胰腺导管细胞株的建立  

Establishment of Pancreatic Ductal Cell Lines with Stably Silencing of MyD88 Mediated by Lentivirus

作　　者：李扬[1,2] 周斌[1,2] 陈珂玲[1,2] 刘勇[2,3] 詹兰[1,2] 李园[1,2,4] 周总光[1,2,3] 

机构地区：[1]四川大学华西医院生物治疗国家重点实验室,成都610041 [2]四川大学华西医院消化外科研究室,成都610041 [3]四川大学华西医院胃肠外科中心,成都610041 [4]四川大学华西医院小儿外科,成都610041

出　　处：《四川大学学报（医学版）》2011年第3期293-297,共5页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(批准号30830100)资助

摘　　要：目的建立稳定沉默髓样分化因子88基因(MyD88)的大鼠胰腺导管细胞株ARIP。方法设计并构建编码MyD88 mRNA的短发卡样RNA(shRNA)质粒表达载体,并用三质粒包装系统包装成慢病毒,分别命名为Lenti-shMyD88-1和Lenti-shMyD88-2。将ARIP分为未处理组、Lenti-Non Target阴性对照组、Lenti-shMyD88-1组和Lenti-shMyD88-2组。并用相应的病毒转导(未处理组只换液)。经流式分选和抗生素筛选结合的方法筛选绿色荧光蛋白阳性细胞,测定细胞的转导效率并用real-ti me PCR方法测定沉默效率。结果成功构建了编码表达MyD88的shRNA表达质粒。经流式分选和抗生素筛选后,慢病毒转导效率均达到100%。与未处理组相比,Lenti-shMyD88-1和Lenti-shMyD88-2对MyD88 mRNA的沉默效率分别为82.73%±1.203%和75.56%±1.176%。结论建立了MyD88基因稳定沉默的胰腺导管细胞株。为探讨MyD88基因与急性胰腺炎的相关性提供了一个全新的、稳定的、可操作的细胞模型。Objective To design and construct a lentiviral vector containing shRNA against rat myeloid differentiation protein 88 gene（MyD88）,and to establish rat pancreatic ductal cell line with stable knockdown of MyD88 expression.Methods Constructed two plasmid expression vectors coding shRNA against MyD88 and converted them into lentiviral particles using three-plamid package system,named as Lenti-shMyD88-1 and Lenti-shMyD88-2.Rat pancreatic ductal cell,ARIP were divided into untreated group,Lenti-Non Target group,Lenti-shMyD88-1 and Lenti-shMyD88-2 treated groups,and transduced with corresponding lentiviral particles.The GFP positive cells were selected by fluorescence-activated cell sorting and puromycin,the MyD88 gene silencing efficiency was detected by real-time RT-PCR.Results After the transduction,we observed highly efficient transduction（reach to 100%） of lentivirus in ARIP cells by fluorescent microscopy and FACS.Quantitative RT-PCR showed that Lenti-shMyD88-1 reduced MyD88 mRNA expression by about 82.73%±1.203% in ARIP cells,and Lenti-shMyD88-2,reduced 75.56%±1.176% as compared with that of the untreated cells（P〈0.05）.Conclusion The results demonstrated that lentivector containing the short hairpin RNA expression cassette specifically targeting MyD88（pLVshMyD88-1,-2） were successfully constructed,which could stably knock down the MyD88 expression in ARIP cells.This study finally provided a new and stable cell model for the study of MyD88＇s function in acute pancreatitis.

关 键 词：髓样分化因子88(MyD88) 慢病毒 RNA干扰 急性胰腺炎 ARIP 

分 类 号：R576[医药卫生—消化系统]