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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: To construct a lentivirus with optimized hTERT promoter and to investigate the specific in vivo imaging of telomerase-positive tumors by using this system in combination with the living animal imaging system. Methods The optimized hTERT promoter was cloned into pGL-Basic plasmid and the optimized hTERT promoter was tested for its function. After determining its driving activity, a third-generation lentivirus expression system containing the promoter and GFP was constructed, and the lentivirus containing the CMV promoter was used as a control. Using Small Animal Fluorescence Imaging System to Study the Role of Optimized hTERT Promoter in Real-Time Tumor Diagnosis in Vivo and in Vivo. Results The promoter function test showed that the hTERT promoter had strict telomerase positive tumor cell specificity and had a high level of reporter gene recombination. The 3rd generation lentiviral packaging system gains high titers of viral particles and efficiently integrates the reporter gene host cells. In vitro studies have shown that in vitro infection of lentivirus with the optimized hTERT promoter specifically in GFP-expressing telomerase positive tumor cells and up to 40 days of reporter gene expression can be maintained. In vivo experiments showed that telomerase positive live tumors were specifically observed in real time after infected with the lentivirus containing the optimized hTERT promoter in 24 h and the in vivo tumor GFP signal was maintained for 30 days in surviving nude mice, And enhanced. Conclusion The optimized hTERT promoter has strict telomerase specificity for the regulation of reporter gene. The third generation lentiviral system can integrate the promoter and reporter gene into the cell genome and achieve telomerase positive tumor cells Specific expression. Combined with small animal live optical imaging system, telomerase positive tumor in nude mice can be noninvasive, real-time and specific imaging, providing a new theoretical basis for the early diagnosis of tumor-specific early diagnosis.