CAJ | 학술논문

目的构建携带胰岛素样生长因子2(IGF2)印迹系统的重组腺病毒,探讨其用于肿瘤靶向治疗的可行性.方法采用聚合酶链反应(PCR)扩增H19 enhancer、DMD以及H19启动子序列,并克隆至pDC-312中；扩增出增强型绿色荧光蛋白(EGFP)和白喉毒素A(DT-A)片段,分别插入到构建好的重组质粒中,构建成重组腺病毒穿梭质粒.重组腺病毒穿梭质粒与Ad5共转染,产生重组腺病毒Ad-EGFP和Ad-DT-A.以Ad-EGFP分别感染正常IGF2印迹(MOI)细胞GES-1和MCF-7以及IGF2印迹丢失(LOI)细胞HCT-8,荧光显微镜下观察各组细胞中EGFP的表达.通过检测腺病毒衣壳蛋白hexon基因的表达证实腺病毒的感染,采用逆转录聚合酶链反应(RT-PCR)和Western blot方法检测Ad-DT-A感染后各组细胞中DT-A基因的表达；采用四甲基偶氮唑蓝(MTT)法和流式细胞术检测Ad-DT-A体外抗肿瘤效应.构建皮下移植瘤裸鼠模型,研究Ad-DT-A在体内的抑瘤作用.结果所构建重组腺病毒Ad-EGFP感染各组细胞后,EGFP蛋白仅在IGF2 LOI细胞HCT-8中表达.各组细胞经Ad-DT-A感染后,DT-A基因仅在HCT-8细胞中表达；且HCT-8细胞以每个细胞10 PFU的Ad-DT-A感染72 h后,其增殖活力降低至(75.4±6.4)％,凋亡率升高至(20.8±5.9)％.Ad-DT-A多点注射入HCT-8移植瘤内,Ad-DT-A能够有效抑制移植瘤的生长,抑瘤率达36.4％.结论成功构建了携带IGF2印迹系统和DT-A基因的重组腺病毒.重组腺病毒Ad-DT-A能够有效杀伤IGF2 LOI肿瘤细胞,为依赖IGF2 LOI的肿瘤靶向治疗开拓了新的途径.
목적 구건휴대이도소양생장인자2(IGF2)인적계통적중조선병독,탐토기용우종류파향치료적가행성.방법 채용취합매련반응(PCR)확증H19 enhancer、DMD이급H19계동자서렬,병극륭지pDC-312중；확증출증강형록색형광단백(EGFP)화백후독소A(DT-A)편단,분별삽입도구건호적중조질립중,구건성중조선병독천사질립.중조선병독천사질립여Ad5공전염,산생중조선병독Ad-EGFP화Ad-DT-A.이Ad-EGFP분별감염정상IGF2인적(MOI)세포GES-1화MCF-7이급IGF2인적주실(LOI)세포HCT-8,형광현미경하관찰각조세포중EGFP적표체.통과검측선병독의각단백hexon기인적표체증실선병독적감염,채용역전록취합매련반응(RT-PCR)화Western blot방법검측Ad-DT-A감염후각조세포중DT-A기인적표체；채용사갑기우담서람(MTT)법화류식세포술검측Ad-DT-A체외항종류효응.구건피하이식류라서모형,연구Ad-DT-A재체내적억류작용.결과 소구건중조선병독Ad-EGFP감염각조세포후,EGFP단백부재IGF2 LOI세포HCT-8중표체.각조세포경Ad-DT-A감염후,DT-A기인부재HCT-8세포중표체；차HCT-8세포이매개세포10 PFU적Ad-DT-A감염72 h후,기증식활력강저지(75.4±6.4)％,조망솔승고지(20.8±5.9)％.Ad-DT-A다점주사입HCT-8이식류내,Ad-DT-A능구유효억제이식류적생장,억류솔체36.4％.결론 성공구건료휴대IGF2인적계통화DT-A기인적중조선병독.중조선병독Ad-DT-A능구유효살상IGF2 LOI종류세포,위의뢰IGF2 LOI적종류파향치료개탁료신적도경.
Objective To explore the feasibility of IGF2 imprinting system in target gene therapy for tumors.Methods The mouse H19 enhancer,DMD and promoter H19 were amplified by PCR from mouse genomic DNA and then cloned into the plasmid pDC312.The EGFP and DT-A fragments were amplified by PCR and cloned into the recombinant plasmid,and then the shuttle plasmid were transfected into HEK293 cells together with the adenoviral vector Ad5,namely,Ad-EGFP and Ad-DT-A.Adenovirus hexon gene expression was applied to confirm the presence of adenovirus infectious.The effect of the IGF2 imprinting system was tested by fluorescence microscopy.RT-PCR and Western blotting after transfection of the recombinant adenoviral vectors into cancer cells were used to show loss of IGF2 imprinting (LOI) and maintenance of IGF2 imprinting ( MOI),respectively.The anti-tumor effect was assessed by MTT and flow cytometry after the HCT-8(LOI).Human breast cancer cell line MCF-7(MOI) and human normal gastric epithelial GES-1 (MOI) cell line were transfected with Ad-DT-A in vitro.The anti-tumor effect was detected by injecting the Ad-DT-A in nude mice carrying HCT-8 tumors.Results The expression of EGFP protein,DT-A mRNA and DT-A protein were seen to be positive only in the HCT-8 tumor cell line.Infection with Ad-DT-A resulted in obviously growth inhibition in HCT-8 cells (75.4 ±6.4)％ compared with that in the control group,and increased the percentage of apoptosis in the HCT-8 cells (20.8 ± 5.9) ％.The antitumor effect was further confirmed by injecting the recombinant adenoviruses in HCT-8 tumor-bearing nude mice,and the results showed that the Ad-DT-A inhibited the tumor growth,with on inhibition rate of 36.4％.Conclusions The recombinant adenoviruses carrying IGF2 imprinting system and DT-A gene have been successfully constructed,while Ad-DT-A can effectively kill the tumor cells showing loss of IGF2 imprinting.It might play an important role in future target gene therapy against malignant tumors based on loss of IGF2 imprinting.