摘要
目的:通过临床前体外实验研究观察溶瘤病毒CNHK300对各种乳腺癌细胞的特异性杀伤作用。方法:通过TRAP-ELISA方法,确认各种乳腺癌细胞和正常成纤维细胞中的端粒酶活性。Western印迹检测腺病毒CNHK300 E1A在端粒酶阳性肿瘤细胞和阴性正常成纤维细胞中的表达差异。通过荧光显微镜观察病毒在细胞中感染和复制的过程。行病毒增殖实验,验证溶瘤病毒CNHK300在端粒酶阳性乳腺癌细胞中选择性复制能力;MTT方法行细胞生长抑制实验,检测CNHK300对端粒酶阳性乳腺癌细胞的选择性杀伤能力。结果:各种乳腺癌细胞MCF-7、BT-549和SK-BR-3的端粒酶均为阳性,MRC-5和BJ细胞的端粒酶均为阴性。CNHK300病毒的E1A可以选择性在端粒酶阳性的乳腺癌细胞株和转染E1A的293细胞中表达,而在端粒酶阴性的正常成纤维细胞株中不表达。CNHK300病毒可以选择性在乳腺癌细胞中复制并杀伤肿瘤细胞,而在正常细胞中复制明显减弱,同时杀伤作用明显下降。结论:本研究证明,hTERT启动子可以用于调控腺病毒E1A选择性在端粒酶阳性肿瘤细胞中表达,并调控病毒在肿瘤细胞中选择性复制,最后产生溶瘤作用。溶瘤病毒可能为肿瘤治疗提供一种新的有力武器。
Objective： To carry out a preclinical study for assessment of the specific killing effect of replication-competent adenovirus CNHK300 on in vitro breast carcinoma cells with various biological status. Methods： Telomerase activity in various breast cancer and normal fibroblast cell lines was confirmed by TRAP-ELISA. Different cells were added with CNHK300 virus and then the E1A protein in the cytoplasm was measured by Western blotting. Fluorescence microscopy was used to observe the CNHK300-EGFP proliferation after the cells were cultured and added with the virus. Virus proliferation assay was applied to evaluate the proliferation selectivity of CNHK300. MTF method was used to evaluate the cytolysis selectivity of CNHK300. Results：The telomerase activity was positive in breast cancer cells MCF-7, BT-549 and SK-BR-3, but not in fibroblast cells. CNHK300 E1A could be detected by Western blotting in telomerase positive breast cancer cells and E1A transformed 293 cells, but not in telomerase negative normal fibroblast cells. CNHK300 replicated in and elimina- ted breast cancer cells efficiently with high specificity in comparison with normal fibroblast cells. Conclusion：The findings in this study suggest that hTERT promoter can be applied to regulate the adenovirus E1A protein expression and to modulate selective replication of the CNHK300 in telomerase-positive breast cancer cells. It may be a potential treatment strategy in cancer.
出处
《军事医学科学院院刊》
CSCD
北大核心
2007年第3期228-231,234,共5页
Bulletin of the Academy of Military Medical Sciences
基金
国家"863"计划重点资助项目(2001AA217031)
国家自然科学基金国际合作项目(30120160824)
