CAJ | 학술논문

目的以单纯疱疹病毒1-胸苷激酶(HSV1-tk)为报告基因,以~(131)I标记的氟-碘阿糖基脲嘧啶(FIAU)为报告探针,对转染HSV1-tk基因的大鼠心肌组织行放射性自显影(ARG)研究,以探讨该报告系统用于心脏报告基因显像的可行性,并为显像的最佳病毒滴度和显像时间提供理论基础.方法制作腺病毒(Ad)为载体携带HSV1-tk基因的重组体Ad5-tk及空载病毒(Ad5-null),SD大鼠心肌内注射Ad5-tk及Ad5-null.根据目的小同进行分组如下:(1)转染报告基因后时间对显像的影响:心肌内转染1×10~8pfu Ad5-tk后第1、2、3、5、7天注射~(131)I-FIAU;(2)转染不同病毒滴度对显像的影响:心肌内注射不同滴度的Ad5-tk(5×10~8,1×10~8,5×10~7,1×10~7pfu),2 d后注射~(131)I-FIAU.对照组均采用注射同体积的Ad5-null.注射~(131)I-FIAU 24 h后断颈法处死大鼠,取出心脏,测量γ计数值,行ARG研究,逆转录聚合酶链反应(RT-PCR)从mRNA水平证实,并对ARG图像和RT-PCR图像行半定量分析.结果 ARG及RT-PCR图像上均可见局部报告基因的表达随感染滴度的增加而增高,随感染时间的延长而降低.半定量分析证实在滴度和时间上RT-PCR及γ计数与放射性自显影呈相关关系:不同滴度Ad5-tk转染大鼠心肌细胞,RT-PCR与ARG相关系数r~2=0.963,P<0.05,γ计数与ARG相关系数r~2=0.996,P<0.01;不同时间Ad5-tk转染大鼠心肌细胞,RT-PCR与ARG相关系数r~2=0.950,P<0.05,γ计数与ARG相关系数r~2=0.980,P<0.01.结论应用ARG方法证实HSV1-tk为报告基因,FIAU为报告探针的系统可以用于心肌报告基因显像,显像最佳的转染滴度为1×10~8pfu,最佳的显像时间为转染后24～48 h,为在体进行报告基因心脏显像提供了理论基础.
목적 이단순포진병독1-흉감격매(HSV1-tk)위보고기인,이~(131)I표기적불-전아당기뇨밀정(FIAU)위보고탐침,대전염HSV1-tk기인적대서심기조직행방사성자현영(ARG)연구,이탐토해보고계통용우심장보고기인현상적가행성,병위현상적최가병독적도화현상시간제공이론기출.방법 제작선병독(Ad)위재체휴대HSV1-tk기인적중조체Ad5-tk급공재병독(Ad5-null),SD대서심기내주사Ad5-tk급Ad5-null.근거목적 소동진행분조여하:(1)전염보고기인후시간대현상적영향:심기내전염1×10~8pfu Ad5-tk후제1、2、3、5、7천주사~(131)I-FIAU;(2)전염불동병독적도대현상적영향:심기내주사불동적도적Ad5-tk(5×10~8,1×10~8,5×10~7,1×10~7pfu),2 d후주사~(131)I-FIAU.대조조균채용주사동체적적Ad5-null.주사~(131)I-FIAU 24 h후단경법처사대서,취출심장,측량γ계수치,행ARG연구,역전록취합매련반응(RT-PCR)종mRNA수평증실,병대ARG도상화RT-PCR도상행반정량분석.결과 ARG급RT-PCR도상상균가견국부보고기인적표체수감염적도적증가이증고,수감염시간적연장이강저.반정량분석증실재적도화시간상RT-PCR급γ계수여방사성자현영정상관관계:불동적도Ad5-tk전염대서심기세포,RT-PCR여ARG상관계수r~2=0.963,P<0.05,γ계수여ARG상관계수r~2=0.996,P<0.01;불동시간Ad5-tk전염대서심기세포,RT-PCR여ARG상관계수r~2=0.950,P<0.05,γ계수여ARG상관계수r~2=0.980,P<0.01.결론 응용ARG방법증실HSV1-tk위보고기인,FIAU위보고탐침적계통가이용우심기보고기인현상,현상최가적전염적도위1×10~8pfu,최가적현상시간위전염후24～48 h,위재체진행보고기인심장현상제공료이론기출.
Objective Radionuclide imaging of reporter gene expression holds promise for noninvasive monitoring of gene therapy. Herpes simplex virus 1 -thymidine kinase ( HSV1-tk ) has been successfully applied to the tumor tissue. We explored the feasibility of the expression imaging of HSV1-tk reporter gene in rat myocardium by using SPECT reporter probe ~(131)-2'-fluoro-2'-deoxy-1-β-D-arabinofuranosyl-5-iodouracil (~(131)I-FIAU) and autoradiography(ARG). Methods The recombinant Ad5-tk carrying HSV1-tk gene and adenovirus (Ad5-null) as vector were constructed and intramyocardlally injected transfection reporter gene : rats were injected with ~(131)I-FIAU at day 1, 2, 3, 5 and 7 after transfection of with various titers of AdS-tk(5×10~8,1×10~8,5×10~7, 1×10~7pfu). After 2 days, rats were injected with ~(131)I-FIAU in tail vein. Equal volume Ad-nulls was intramyocardially injected to control rats. Rats were killed 24 h after injection of ~(131)I-FIAU and the hearts were rapidly dissected for gamma counts measurement. The total myocardial ~(131)I-FIAU accumulation was quantified in percent of injected dose per gram myocardium (% ID/g). The myocardial reporter gene expression was semi-quantitatively determined by ARG and RT-PCR. Results ARG and RT-PCR showed that the local expression of reporter gene increased in proportion with increasing titer and decreased in proportion with time post injection. The semi-quantitative assay showed there were significant correlations among % ID/g, RT-PCR and ARG: r~2 = 0.963, P<0.05 for RT-PCR and ARG; r~2=0.996, P<0.01 for % ID/g and ARG in rats received various reporter gene titers at identical time point post injection ; r~2=0.950, P<0.05 for RT-PCR and ARG; r~2=0.980, P<0.01 for % ID/g and ARG for rats received identical reporter gene titer on various time points post injection. Conclusions The present study showed that cardiac reporter gene imaging with HSV1-tk as reporter gene and ~(131)I-FIAU as reporter probe was feasible in rats. The optimal Ad5-tk titer is 1×10~8 pfu and the optimal imaging time is 24 h to 48 h post gene transfer. HSV1-tk/FIAU may be used for the noninvasive monitoring of cardiac gene therapy.