CAJ | 학술논문

目的利用共表达的分泌型荧光素酶Gluc(gaussia princeps luciferase)和非分泌型荧光素酶Fluc(firefly luciferase)研究重组8型腺相关病毒(recombinant adeno-associated virus type 8,rAAV8)介导的转基因在小鼠体内的表达特点.方法制备携带双荧光素酶基因的重组8型腺相关病毒rAAV8-Gluc/Fluc,体外感染HEK293细胞并检测上清和胞内Gluc和Fluc活性；将不同剂量的rAAV8-Gluc/Fluc尾静脉注射或肌内注射至BALB/c小鼠,通过尾静脉采血检测Gluc活性,通过活体成像和裂解组织检测Fluc活性.结果成功制备了rAAV8-Gluc/Fluc,可以有效感染HEK293细胞,同时分泌表达Gluc和胞内表达Fluc；尾静脉注射或肌内注射rAAV8-Gluc/Fluc至小鼠后,外周血Gluc活性均在注射后10 ～20 d达到高峰并稳定持续120 d以上,Gluc活性随注射剂量增加而增高；静脉注射rAAV8-Gluc/Fluc时Fluc主要在肝脏表达,在骨骼肌和心肌有少量表达,而肌内注射时Fluc既在肌内注射局部表达同时也在肝脏中表达.结论本研究成功制备了携带双荧光素酶基因rAAV8-Gluc/Fluc,研究了其介导的转基因在小鼠体内的表达特点,为rAAV8的临床前应用打下基础.
목적 이용공표체적분비형형광소매Gluc(gaussia princeps luciferase)화비분비형형광소매Fluc(firefly luciferase)연구중조8형선상관병독(recombinant adeno-associated virus type 8,rAAV8)개도적전기인재소서체내적표체특점.방법 제비휴대쌍형광소매기인적중조8형선상관병독rAAV8-Gluc/Fluc,체외감염HEK293세포병검측상청화포내Gluc화Fluc활성；장불동제량적rAAV8-Gluc/Fluc미정맥주사혹기내주사지BALB/c소서,통과미정맥채혈검측Gluc활성,통과활체성상화렬해조직검측Fluc활성.결과 성공제비료rAAV8-Gluc/Fluc,가이유효감염HEK293세포,동시분비표체Gluc화포내표체Fluc；미정맥주사혹기내주사rAAV8-Gluc/Fluc지소서후,외주혈Gluc활성균재주사후10 ～20 d체도고봉병은정지속120 d이상,Gluc활성수주사제량증가이증고；정맥주사rAAV8-Gluc/Fluc시Fluc주요재간장표체,재골격기화심기유소량표체,이기내주사시Fluc기재기내주사국부표체동시야재간장중표체.결론 본연구성공제비료휴대쌍형광소매기인rAAV8-Gluc/Fluc,연구료기개도적전기인재소서체내적표체특점,위rAAV8적림상전응용타하기출.
Objective Recombinant adeno-associated virus type 8 (rAAV8) mediating transgene expression in mice was investigated using co-expressed report gene of secreted Gaussia princeps luciferase (Gluc) and non-secreted firefly luciferase(Fluc).Methods rAAV8-Gluc/Fluc was prepared and infected HEK293 cells to test its performance in vitro.BALB/c mice were received rAAV8-Gluc/Fluc at different doses by intravenous injection (iv) or intramuscular injection (im).Then Gluc activities in blood were measured,the whole-body images for Fluc activities were performed and Fluc activities of tissue lysate were also detected.Results rAAV8-Gluc/Fluc was successfully prepared and could infected HEK293 cells.The Gluc was mainly detected in the culture media while the Fluc was mainly detected within cells.The blood Gluc activities of mice received rAAV8-Gluc/Fluc by iv or im peaked at 10-20 d post injection and persisted for at least 120d.The blood Gluc activities increased at the rAAV8-Gluc/Fluc dose-dependent manner.For mice received rAAV8 by iv,Fluc mainly expressed in liver and minor Fluc expression was also found in cardiac muscle and skeletal muscle.For mice received rAAV8-Gluc/Fluc by im,Fluc mainly expressed in local skeletal muscle and secondly in liver.Conclusion rAAV8-Gluc/Fluc has been prepared successfully and its mediating transgene expression in mice has been investigated.This research will facilitate preclinical studies for rAAV8-mediated gene therapy.