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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 observe the specific killing effect of oncolytic virus CNHK300 on various breast cancer cells by preclinical in vitro experiments. Methods: Telomerase activity in various breast cancer cells and normal fibroblasts was confirmed by TRAP-ELISA. The difference of expression of adenovirus CNHK300 E1A between telomerase positive tumor cells and negative normal fibroblasts was detected by Western blotting. Fluorescence microscopy was used to observe the process of virus infection and replication in the cells. The virus proliferation assay was used to verify the selective replication ability of oncolytic virus CNHK300 in telomerase positive breast cancer cells. MTT assay was used to detect the cytotoxicity of CNHK300 on telomerase positive breast cancer cells. Results: The telomerase activity of breast cancer cells MCF-7, BT-549 and SK-BR-3 were all positive. The telomerase activity of MRC-5 and BJ cells was negative. E1A of the CNHK300 virus can be selectively expressed in telomerase-positive breast cancer cells and E1A-transfected 293 cells, but not in telomerase-negative normal fibroblast strains. CNHK300 virus can selectively replicate and kill tumor cells in breast cancer cells, whereas in normal cells, the replication is obviously weakened and the killing effect is obviously decreased. Conclusion: This study demonstrates that hTERT promoter can be used to regulate the expression of adenovirus E1A selectively in telomerase positive tumor cells and regulate the selective replication of the virus in tumor cells, finally resulting in oncolytic effect. Oncolytic virus may provide a new powerful weapon for cancer treatment.