关键词: 短发卡RNA, 慢病毒载体, CHOP, 细胞凋亡, 内质网应激

Abstract: Objective We construct an effective eukaryotic expression vector of short hairpin RNAagainstmouse C/EBPhomologous protein, and study the effect on apoptosis of Heap 1-6 cells when silencing of CHOP. Methods We designed and synthesized shRNA target sequence and non-homology shRNA sequence as negative control, which were recombinantd into pLKO.1-TRC vector after annealing. Then products of transformation and amplificationwere indentified by rapid double enzyme digestion.Heap 1-6 cells were transfected with viral supernatant from 293T cells transfected with the lentiviral packaging method. A stable strain was obtained from 10 days of continuous screening with a culture medium containing adenine. We detected the protein expressions of caspase9 and c-PARP by western blot and analysed cell cycles by flow cytometry. Results The results showed that shRNA lentiviral vectors were constructed successfully. Compared with the negative group, the shCHOP group could significantly inhibit the expression of CHOP protein and reduced the apoptosis induced by tunicamycin (p<0.05). Conclusion CHOP gene silencing could down-regulated the apoptosis of Heap 1-6 cells induced by endoplasmic reticulum stress.

Key words: short hairpin RNA, lentiviral vector, C/EBP-homologous protein, apoptosis, endoplasmic reticulum stress