Clinical effect of ultrasound-targeted microbubbles loaded with cytidine deaminase-thymidine kinase double suicide gene in treatment of lung cancer

Abstract:

Objective：To prepare the ultrasound microbubbles loaded with the cytidine deaminase-thymidine kinase（CD-TK） double suicide gene，and to investigate its killing effect on lung cancer. Methods：The microbubbles loaded with the CD-TK double suicide gene were prepared，and their morphology and size were observed. Propidium iodide was used for the staining of plasmids to observe fluorescence and calculate the binding rate of microbubbles and genes. Lung cancer H1299 cells were cultured and a nude mouse model of lung cancer was established. The cells were randomly divided into control group，ultrasound（U）+microbubble（M）+CD-TK group，U+M+CD-TK+5-fluorcytosine（5-FC） group，U+M+CD-TK+ganciclovir（GCV） group，and U+M+CD-TK+5-FC+GCV group，with 1×107 cells in each group. The lung cancer H1299 cells were transfected with the microbubbles，and after 24 hours，the expression of green fluorescent protein（GFP） was observed and the effect of GFP transfection and 24-hour apoptosis and cell cycle were measured. In the animal experiment in vivo，there were 6 nude mice in each group；the mice in the control group were treated with the injection of 200 μL phosphate-buffered saline，and those in all the other groups were given the tail vein injection of an equal volume of the microbubbles loaded with the CD-TK double suicide gene. The development of microbubbles was observed，and gene transfec－tion was performed at the tumor site by ultrasound irradiation. Intraperitoneal injection of 5-FC and GCV was performed for one week. The longest and shortest diameters of the tumors of each nude mouse were recorded every week，and the change in tumor volume was calculated and recorded. Tumor samples were collected for the nude mice at week 5；immunofluorescence assay was used to measure the expression of TdT（the TUNEL method） and proliferating cell nuclear antigen（PCNA），and cell apoptosis and proliferation in tumor tissue were assessed. Results：The microbubbles loaded with the CD-TK double suicide gene were successfully prepared. 5-FC and GCV combined with ultra－sound microbubbles loaded with CD-TK double suicide gene had a marked therapeutic effect and significantly promoted apoptosis[an apoptosis rate of (28.45±1.75）%] and inhibited proliferation[（61.40±4.31）% cells in S phase），with a significantly better effect than the control group（P=0.000），the 5-FC group（P=0.002），and the GCV group（P=0.012）. The U+M+CD-TK+5-FC+GCV group had the lowest tumor volume and weight，the highest number of apoptotic cells，and the most significant inhibition of proliferation. Conclusion：5-FC and GCV combined with ultrasound microbubbles loaded with the CD-TK double suicide gene can promote the apoptosis and inhibit the proliferation of lung cancer cells and thus have a marked killing effect on lung cancer.