携带IL-24的溶瘤腺病毒作用于乳腺癌的体外实验研究

目的 构建携带IL-24基因的溶瘤腺病毒CNHK600-IL-24,评估CNHK600-IL-24在体外对乳腺癌细胞的杀伤能力.方法 将IL-24基因插入腺病毒穿梭载体SG502-ΔCR2,与5型腺病毒骨架载体pPE3共转染至人胚肾293细胞,获得溶瘤腺病毒CNHK600-IL-24.荧光显微镜观测及病毒体外增殖实验测...

The in vitro study on the infection of oncolytic adenovirus CNHK600-IL-24 to breast cancer cells

Objective To construct an oncolytic adenovirus CNHK600-IL-24, and to evaluate its tumor-specific proliferation and killing capacity in vitro. Methods The IL-24 gene was cloned into adenovirus shuttle vector SG502-ΔCR2, and CNHK600-IL-24 was obtained by cotransfection of SG502-INS-IL-24 and pPE3 plasmids and subsequent recombination in 293 cells. Fluorescence microscopy and viral replication experiments were performed to evaluate the proliferation ability in breast cancer cell line MDA-MB-231 and normal fibroblastic cell line MRC-5. MTT assay was performed to determine the inhibitory effect on the two cell lines. ELISA was performed to detect IL-24 in the supernatant after the virus infection. And we detected the intracellular IL-24 protein by Western blot analysis. Finally, the flow cytometry was used to detect cell apoptosis. Results The oncolytic adenovirus CNHK600-IL-24 was correctly constructed and confirmed by restriction DNA sequence analysis and PCR. The titer of CNHK600-IL-24 reached 1.9×10¹⁰ pfu/mL. CNHK600-IL-24 proliferated and replicated more efficiently in MDA-MB-231 cells than in MRC-5 cells. CNHK600-IL-24 could kill MDA-MB-231 cells at very low MOI, while the killing capacity to MRC-5 cells was rather weak. The amount of IL-24 increased obviously 48 hours postinfection in MDA-MB-231 cells, but for MRC-5 cells, the level of IL-24 maintained low. The flow cytometry analysis showed that CNHK600-IL-24 induced apoptosis in the MDA-MB-231 cells, but the apoptosis of MRC-5 cells was low. Conclusions The high-titer oncolytic adenovirus CNHK600-IL-24 was successfully constructed and purified. CNHK600-IL-24 could proliferate specifically in breast cancer cells and lysed them.