摘要
目的构建一种含优化型人端粒酶催化亚单位(hTERT)启动子的慢病毒,结合小动物活体光学成像系统,研究该启动子对端粒酶阳性肿瘤的特异性活体成像作用。方法将文献报道的优化型hTERT启动子克隆到pGL-Basic质粒,对优化型hTERT启动子进行功能检测。确定其驱动活性之后,构建一种含该启动子和GFP的第3代慢病毒表达系统,以含CMV启动子的慢病毒作为对照。采用小动物荧光成像系统体内外研究优化型hTERT启动子在肿瘤实时诊断中的作用。结果启动子功能检测发现hTERT启动子具有严格的端粒酶阳性肿瘤细胞特异性,并且具有很高的报告基因报答水平。第3代慢病毒包装系统获得了高滴度的病毒颗粒并且能高效整合报告基因之宿主细胞。体外研究表明,含有优化型hTERT启动子的慢病毒体外感染细胞后,能够在端粒酶阳性肿瘤细胞内特异表达GFP,并且能维持长达40d的报告基因表达时间。体内实验发现,感染含优化型hTERT启动子的慢病毒之后24h,端粒酶阳性活体肿瘤能够被特异性的实时观察到,并且肿瘤内的GFP信号在存活裸鼠体内维持30d,随肿瘤增大而增强。结论优化后hTERT启动子对报告基因的调控具有严格的端粒酶特异性,第3代慢病毒系统可将该启动子和报告基因整合至细胞基因组,并实现在端粒酶阳性肿瘤细胞内的特异表达。结合小动物活体光学成像系统,荷瘤裸鼠内的端粒酶阳性肿瘤能够被无创的、实时的、特异性的成像,为肿瘤的特异性早期诊断提供新的实验理论基础。
Objective The purpose of the study was to create a tumor specific lentivirus that contain human telomerase reverse transcriptase promoter. Combined with whole-body optical imag- ing equipment, this hTERT promoter was evaluated for specific in vivo imaging of telomerase-posi- tive tumors. Methods A formerly reported optimized hTERT promoter was subcloned into a pGL3 -basic plasmid, and the promoter function was tested by luciferase assay. After the activity of pro- moter was confirmed, a third generation lentiviral system that contains this promoter and GFP was constructed, along with a controlled lentivirus that contained CMV promoter. In vitro and in vivo imaging experiments were conducted to study the optimized promoter on real - time tumor diagnosis Results Promoter function test indicated that this hTERT promoter was strictly specific to telomerase-positive tumor cells, showing high level of reporter gene expression in these cells. The third generation lentivirus package system achieved high titer lentivirus and it was able to inte- grate target gene into host cell genome. In vitro experiment indicated that transfection of cells by lentivirus containing optimized hTERT promoter induced GFP expression specifically in telom- erase-positive tumor cells, lasting for 40 days. In vivo study indicated that 24 hours after being transfected with lentivirus containing optimized hTERT promoter, telomerase-positive tumors were real-time specifically observed by in vivo imaging system. And GFP signal in tumors lasted for 30 days, with scope and intensity increasing as tumor grows. Conclusion Optimized hTERT promoter is strictly telomerase specific for expressing reporter gene. The third generation lentivirus system is able to integrate hTERT promoter and GFP into cell genome and achieve specific expression in telomerase positive tumor cells. Combined with small animal in vivo imaging system, telomerase positive tumors on nude mice can be noninvasively, real time and specifically imaged. It provided novel laboratory theory for early stage tumor specific diagnosis.
出处
《实用临床医药杂志》
CAS
2010年第3期4-12,共9页
Journal of Clinical Medicine in Practice
基金
国家自然科学基金资助项目(30871150)
重庆市自然科学基金资助项目(CSTC
2008BB5274)
