CAJ | 학술논문

目的构建poly(A)-Promoter介导的携带人肿瘤生长抑制因子4(ING4)和人白细胞介素-24(IL-24)双基因的重组腺病毒共表达载体Ad-ING4-poly(A)-Promoter-IL-24(简称Ad-ING4-IL-24),研究Ad-ING4-IL-24对HepG-2人肝癌细胞生长的影响.方法以含有poly(A)和Promoter (hEF1-eIF4g)基因片段的pORF-mbcl-2α质粒为模板,PCR扩增poly(A)-Promoter目的片段(含Sal Ⅰ,Not Ⅰ),亚克隆至pAdTrack-CMV载体中以构建pAdTrack-CMV-poly(A)-Promoter空载体,再分别以pcDNA3.0-ING4和pcDNA3.0-IL-24重组质粒为模板,PCR扩增ING4(含BglⅡ,Sal Ⅰ)和IL-24(含XhoⅠ,XbaⅠ)目的基因片段,并依次插入pAdTrack-CMV-poly(A)-Promoter载体中构建pAdTrackCMV-ING4-Poly(A)-Promoter-IL-24双基因共表达重组转移载体,测序正确后用Pme Ⅰ酶线性化,与腺病毒骨架质粒pAdEasy-1构建同源重组腺病毒质粒pAdEasy-1-pAdTrack-CMV-ING4-poly(A)-Promoter-IL-24,再经Pac Ⅰ酶线性化后用脂质体转染QBI-293A包装细胞,经多轮扩增后收获Ad-ING4-IL-24重组腺病毒子,其效价可达3.5×109PFU/ml,用RT-PCR和Western blot法分别鉴定ING,4和IL-24基因在HepG-2人肝癌细胞中的转录和表达,MTT法和流式细胞仪检测Ad-ING4-IL-24对HepG-2人肝癌细胞生长抑制和诱导细胞凋亡的功能及其增效作用.结果 DNA测序结果显示pAdTrack-CMV转移载体中插入的ING4、poly(A)-Promoter和IL-24序列与GenBank报道的完全一致,RT-PCR和Western blot检测结果显示Ad-ING4-IL-24能成功介导ING4和IL-24基因在HepG-2人肝癌细胞中表达;并能明显抑制HepG-2人肝癌细胞的生长和诱导其凋亡,其中Ad-ING4-IL-24双基因组更优于相应各单基因组.结论成功构建了poly(A)-Promoter介导的Ad-ING4-IL-24双基因共表达重组腺病毒载体,Ad-ING4-IL-24不仅能明显抑制HepG-2人肝癌细胞生长和诱导其凋亡,而且与Ad-ING4和AdIL-24单基因组相比具有抑癌增效作用.
목적 구건poly(A)-Promoter개도적휴대인종류생장억제인자4(ING4)화인백세포개소-24(IL-24)쌍기인적중조선병독공표체재체Ad-ING4-poly(A)-Promoter-IL-24(간칭Ad-ING4-IL-24),연구Ad-ING4-IL-24대HepG-2인간암세포생장적영향.방법 이함유poly(A)화Promoter (hEF1-eIF4g)기인편단적pORF-mbcl-2α질립위모판,PCR확증poly(A)-Promoter목적 편단(함Sal Ⅰ,Not Ⅰ),아극륭지pAdTrack-CMV재체중이구건pAdTrack-CMV-poly(A)-Promoter공재체,재분별이pcDNA3.0-ING4화pcDNA3.0-IL-24중조질립위모판,PCR확증ING4(함BglⅡ,Sal Ⅰ)화IL-24(함XhoⅠ,XbaⅠ)목적 기인편단,병의차삽입pAdTrack-CMV-poly(A)-Promoter재체중구건pAdTrackCMV-ING4-Poly(A)-Promoter-IL-24쌍기인공표체중조전이재체,측서정학후용Pme Ⅰ매선성화,여선병독골가질립pAdEasy-1구건동원중조선병독질립pAdEasy-1-pAdTrack-CMV-ING4-poly(A)-Promoter-IL-24,재경Pac Ⅰ매선성화후용지질체전염QBI-293A포장세포,경다륜확증후수획Ad-ING4-IL-24중조선병독자,기효개가체3.5×109PFU/ml,용RT-PCR화Western blot법분별감정ING,4화IL-24기인재HepG-2인간암세포중적전록화표체,MTT법화류식세포의검측Ad-ING4-IL-24대HepG-2인간암세포생장억제화유도세포조망적공능급기증효작용.결과 DNA측서결과현시pAdTrack-CMV전이재체중삽입적ING4、poly(A)-Promoter화IL-24서렬여GenBank보도적완전일치,RT-PCR화Western blot검측결과현시Ad-ING4-IL-24능성공개도ING4화IL-24기인재HepG-2인간암세포중표체;병능명현억제HepG-2인간암세포적생장화유도기조망,기중Ad-ING4-IL-24쌍기인조경우우상응각단기인조.결론 성공구건료poly(A)-Promoter개도적Ad-ING4-IL-24쌍기인공표체중조선병독재체,Ad-ING4-IL-24불부능명현억제HepG-2인간암세포생장화유도기조망,이차여Ad-ING4화AdIL-24단기인조상비구유억암증효작용.
Objective To construct a recombinant adenoviral vector carrying and co-expressing human inhibitor of growth 4(ING4) and human interleukin-24(IL-24) mediated by poly( A)-Promoter[Ad-ING4-poly(A)-Promoter-IL-24, referred to as Ad-ING4-IL-24] and explore its effect on the growth of HepG-2 human hepatocellular carcinoma cellsin vitro. Methods The poly(A)-Promoter(hEFl-elF4g) (Sal Ⅰ and Not Ⅰ ), ING4 ( Bgl Ⅱ and Sal Ⅰ ), and IL-24 ( Xho Ⅰ and Xba Ⅰ ) fragments were amplified by PCRusing pORF-mbcl-2α, pcDNA3.0-IL-24, and pcDNA3.0-ING4 plasmids as templates and subcloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-ING4-poly (A)-Promoter-lL-24, respectively. The pAdTrack-CMV-ING4-poly (A)-Promoter-IL-24 transfer vector linearized with Pme Ⅰ digestion and pAdEasy-1 backbone vector was further cotransformed into the bacteria BJ5183 competent cells for homologous recombination. The resultant pAdEasy-l-pAdTrack-CMV-ING4-poly ( A )-Promoter-IL-24 homologous recombinant plasmids were linearized with Pac Ⅰ digestion and transfected into the human embryonic kidney 293 (QBI-293A) cells by liposome, leading to formation of the recombinant adenoviruses Ad-ING4-IL-24co-expressing ING4 and IL-24. The Ad-ING4-IL-24 were amplified in QBI-293A cells and its titer was up to 3.5 × 109 PFU/ml. Adenovirus-mediated ING4 and IL-24 genes expression in HepG-2 cells was examined by RT-PCR and Western blot. The growth-suppressing and apoptosis-inducingg effect of Ad-ING4-IL-24 coexpressing ING4 and IL-24 on HepG-2 human hepatocellular carcinoma cells was assessed by MTT assay and FCM, respectively. Results DNA sequencing showed that the ING4, poly (A)-Promoter, and IL-24 fragments subcloned into pAdTrack-CMV plasmids were completely identical to those reported in GenBank.ING4 and IL-24 gene mediated by adenovirus could both successfully express in HepG-2 cells. Adenovirusmediated ING4 and IL-24 co-expression significantly suppressed HepG-2 hepatocellular carcinoma cell growth and induced cell apoptosis, and the effect of Ad-ING4-IL-24 group was more significant than AdING4 and Ad-IL-24 group. Conclusion The adenoviral vector co-expressing ING4 and IL-24 mediated by poly(A)-Promoter(Ad-ING4-IL-24) was successfully constructed. Ad-ING4-IL-24 had marked anti-tumor effect in suppressing HepG-2 human hepatocellular carcinoma cell growth and inducing cell apoptosis in vitro. Compared with Ad-ING4 and Ad- IL-24, Ad-ING4-IL-24 enhanced anti-tumor effect.