MafA逆转录病毒表达载体的构建及其在肝原始细胞中的稳定表达

Construction of MafA recombinant retroviral vector and its stable expression in liver epithelial progenitor cells

目的:构建表达MafA(v-maf musculoaponeurotic fibrosarcoma oncogene homologue A)的逆转录病毒表达载体,并建立稳定表达MafA的肝原始细胞系(liver epithelial progenitor cells,LEPCs)。方法:通过PCR方法克隆MafA基因全长.将其构建到pBMN—Z—IRES-Neo逆转录病毒载体中获得pBMN—MafA—Neo载体;将该载体导入Phoenix包装细胞系,收集病毒上清并感染LEPCs,筛选稳定表达MafA的LEPCs;RT-PCR方法检测MafA表达对LEPCs分子表型的影响。结杲:成功构建pBMN—MafA-Neo载体,并获得稳定表达MafA基因的肝原始细胞系(LEPCs—MafA)。LEPCs-MafA细胞GK和GLUT2基因表达高于LEPCs。结论:成功获得稳定表达MafA的肝原始细胞系,为研究MafA诱导肝干细胞向胰腺细胞转分化奠定了基础。

Objective： To construct a recombinant retroviral vector harboring MafA and to establish a liver epithelial progenitor cell （LEPC） line for stable expression of MafA. Methods： MafA was amplified by PCR and suncloned into pBMN-ZIRES-Neo vector to obtain pBMH-MafA-Neo vector. After introducing the pBMN-MafA-Neo into Phoenix package cells,the cell culture supernatant was used to infect LEPCs. LEPCs stably expressing MafA gene were screened out. RT-PCR was used to detect the influence of MafA on the phenotype of LEPCs. Results： We successfully constructed pBMH-MafA-Neo vector and obtained LEPCs which stably expressed MafA. Expression of GK and GLUT2 genes in LEPCs-MafA cells was higher than that in the LEPCs. Conclusion： We have successfully obtained LEPCs which can stably express MafA,laying a basis for studying the differentiation of LEPCs into pancreas cells.