摘要目的 采用RNA干扰技术沉默大鼠胰腺腺泡细胞AR42J的Beclin1基因表达,构建稳定的Beclin1基因沉默的AR42J细胞系.方法 设计并合成3种靶向大鼠Beclin1 mRNA的shRNA及阴性对照shRNA,分别插入质粒GV112,重组质粒分别命名为p-sh-Beclin1-1、p-sh-Beclin1-2、p-sh-Beclin1-3 及p-shRNA-NC.应用lipo3000将重组质粒瞬转入AR42J细胞,应用RT-PCR检测细胞Beclin1 mRNA表达,筛选出沉默效率最高的质粒,在293T细胞内经慢病毒包装(LV-shBeclin1),感染AR42J细胞,应用嘌呤霉素筛选,采用RT-PCR及蛋白质印迹法分别检测病毒感染细胞Beclin1 mRNA和蛋白表达.结果 重组质粒经琼脂糖凝胶电泳分离及测序证实shRNA序列符合预期.p-sh-Beclin1-1、p-sh-Beclin1-2、p-sh-Beclin1-3及p-shRNA-NC转染后AR42J细胞Beclin1 mRNA表达抑制率分别为(17.8±4.0)％、(30.6±2.8)％、(45.8±7.7)％、(7.0±11.8)％.3个p-sh-Beclin1转染细胞的表达抑制率均较未转染细胞显著增加,差异有统计学意义(P值均＜0.05),而转染p-shRNA-NC细胞的表达抑制率与未转染细胞的差异无统计学意义.抑制率最高的p-sh-Beclin1-3经慢病毒包装,感染AR42J细胞,感染细胞的BeclinmRNA表达抑制率为(86.1±1.2)％,蛋白表达抑制率为(87.9±2.8)％,与未感染细胞的差异均有统计学意义(P值均＜0.01).结论 成功建立Beclin1基因沉默的AR42J细胞株,为探讨Beclin1基因在急性胰腺炎发病机制中的作用提供新的细胞模型.

abstractsObjective To silence the beclin1 gene expression by using RNA interference technology in AR42J rat pancreatic acinar cells,and build a stable AR42J line silencing beclin1.Methods Three kinds of shRNA targeting rat beclin1 mRNA and negative control shRNA were designed and synthesized,and were inserted into the plasmids GV112,respectively.The recombinant plasmids were named as p-sh-Beclin1-1,psh-Beclin1-2,p-sh-Beclin1-3 and p-shRNA-NC.Lipo3000 was used to transfect the recombinant plasmid into AR42J cells,the expression of beclin1 mRNA were detected by RT-PCR to screen for the most efficient silencing plasmid,and then it was packaged into lentiviral (LV).AR42J cells were infected with LV and screened by puromycin.Beclin1 mRNA and protein expression was determined by RT-PCR and Western blot.Results The recombinant plasmid was confirmed by agarose gel electrophoresis and sequencing showed that shRNA sequences were in line with expectations.The beclin1 mRNA inhibition rates of AR42J cells after p-sh-Beclin1-1,p-sh-Beclin1-2,p-sh-Beclin1-3 and p-shRNA-NC transfection were (17.8 ± 4.0) ％,(30.6 ± 2.8) ％,(45.8 ± 7.7) ％,(7.0 ± 11.8) ％,respectively.The inhibition rates of three p-sh-Beclin1 transfection cells were significantly higher than that in non-transfection cells,and the difference was statistically significant (P ＜ 0.05).While the inhibition rate of p-shRNA-NC transfection cells was not significantly different from that of non-transfection cells,p-sh-Beclin 1-3 with highest rate of inhibition was packaged by LV,and infected AR42J cells,then puromycin was applied to screen,inhibition rate of beclin mRNA expression in LV infection cells was (86.1 ± 1.2) ％,and the protein expression inhibition rate was (87.9 ± 2.8) ％,and the difference between infection and non-infection groups was statistically significant (P ＜ 0.05).Conclusions The stable AR42J line silencing beclin1 is successfully established,which can provide a new cell model for future research of the role of beclin1 in the pathogenesis of acute pancreatitis.