CAJ | 학술논문

目的探讨携带凋亡素基因的重组腺相关病毒构建方法,观察其体外抑制膀胱癌的效应.方法构建重组质粒pAAV-VP3,经EcoRI/Sal Ⅰ双酶切鉴定和基因测序无误后,采用三质粒共转染法包装重组腺相关病毒rAAV-VP3.收获病毒后聚合酶链反应(PCR)扩增病毒液中的目的基因鉴定重组病毒,对其进行纯化后透射电镜观察病毒颗粒,并检测病毒滴度.按每个细胞5×105个载体基因组的剂量感染EJ细胞后,逆转录(RT) -PCR检测VP3基因在EJ细胞中的转录,Western blot法检测凋亡素蛋白的表达.透射电镜扫描观察感染重组病毒后肿瘤细胞的超微结构变化,流式细胞术(FCM)检测重组病毒对EJ细胞的影响.结果转染72 h后重组腺相关病毒rAAV-VP3包装成功,滴度为5.1×1011个载体基因组/ml,电镜下可见病毒颗粒.感染EJ细胞后,可检测到VP3基因的转录和凋亡素蛋白表达,电镜观察到凋亡形态学改变,FCM检测S期细胞比例降低和G2/M期细胞比例增高,与对照组比较差异有统计学意义(P＜0.01).结论重组腺相关病毒rAAV-VP3在体外能够发挥生物学活性,其介导的凋亡素表达抑制膀胱癌的体外效应为体内实验奠定了基础.
목적 탐토휴대조망소기인적중조선상관병독구건방법,관찰기체외억제방광암적효응.방법 구건중조질립pAAV-VP3,경EcoRI/Sal Ⅰ쌍매절감정화기인측서무오후,채용삼질립공전염법포장중조선상관병독rAAV-VP3.수획병독후취합매련반응(PCR)확증병독액중적목적기인감정중조병독,대기진행순화후투사전경관찰병독과립,병검측병독적도.안매개세포5×105개재체기인조적제량감염EJ세포후,역전록(RT) -PCR검측VP3기인재EJ세포중적전록,Western blot법검측조망소단백적표체.투사전경소묘관찰감염중조병독후종류세포적초미결구변화,류식세포술(FCM)검측중조병독대EJ세포적영향.결과 전염72 h후중조선상관병독rAAV-VP3포장성공,적도위5.1×1011개재체기인조/ml,전경하가견병독과립.감염EJ세포후,가검측도VP3기인적전록화조망소단백표체,전경관찰도조망형태학개변,FCM검측S기세포비례강저화G2/M기세포비례증고,여대조조비교차이유통계학의의(P＜0.01).결론 중조선상관병독rAAV-VP3재체외능구발휘생물학활성,기개도적조망소표체억제방광암적체외효응위체내실험전정료기출.
Objective To construct the recombinant adeno-associated virus (AAV) expressing apoptin and investigate the anti-tumor capabilities of AAV-mediated expression of VP3 to human bladder cancer cells in vitro.Methods In this study the AAV Helper-Free System was used to generate the recombinant adeno-associated virus expressing VP3 gene.The VP3 gene was cloned into the expression vector pAAV-MCS and the recombinant plasmid pAAV-VP3 was confirmed by double enzymatic digestion using EcoR Ⅰ and Sal Ⅰ.The recombinant expression plasmid was co-transfected into the AAV-293 cells with pHelper and pAAV-RC to produce recombinant AAV viral particles.The morphological features of the virus particles were examined under the electron microscopy.The physical titer of recombinant AAV was measured through digoxigenin-labeled CMV probe dot blot method,and the recombinant virus was verified by polymerase chain reaction (PCR) of the exogenous interest genes of apoptin.EJ cells were infected by recombinant virus with 5 × 105 vector genomes per cell.Reverse transcription ( RT)-PCR and Western blotting were performed to detect the transcription and expression of apoptin.Concerning on the anti-tumor activity in vitro,transmission electron microscopy was used to determine the apoptotic morphological changes of EJ cells after rAAV-VP3 infection.The cell cycle and apoptosis of EJ cells were quantified by using flow cytometry at 4th day after infection of rAAV-VP3.Results The expression vector pAAV-VP3 was successfully constructed and the sequence of the VP3 was confirmed by DNA sequencing.The rAAV-VP3 was obtained by three plasmids cotransfection into AAV-293 cells after 72 h.The recombinant adeno-associated virus had a high titer of 5.1 × 1011 v.g./ml,and virus particles could be observed under the electron microscopy.After infection of EJ cells with rAAV-VP3,the transcription and expression of VP3 gene were detected by RT-PCR and Western blotting.The rAAV-VP3-induced morphological changes of apoptosis such as fragmentation and apoptotic body were observed under the transmission electron microcopy.Moreover,the proliferation of EJ cells was slowed down,and the proportion of cells at S phase was decreased and that at G2/M phase was blocked after rAAV-VP3 infection as compared with control group (P ＜0.01 ).Conclusion AAV-mediated VP3 gene transfer may be a new therapeutic technique for the treatment of bladder neoplasms,which may lay a foundation for further studies on anti-tumor effect of bladder cancer in vivo.