Ultrasound parameters and transfection efficiency of microbubble carrying suicide gene for transfecting ovarian cancer cell lines SKOV_3
AIM: To investigate the ultrasound parameters and transfection efficiency of microbubble carrying herpes simplex virus thymidine kinase suicide gene (hs_1wtk) for transfecting SKOV_3 cells mediated by the ultrasound. METHODS: Under the combination conditions of different ultrasound exposure times ( 8,15,30,and 60 s with the interval as 1 s) and microbubbles at different concentrations, SKOV_3 cells were exposed to the ultrasound so as to screen out the optimal ultrasound exposure time and microbubble concentration by 3- ( 4,5-dimethyhhizol-2-y1 ) 2,5-diphenyl tetrazolium bromide(MTT) assays. The SKOV_3 cells were divided into 6 groups and treated by ultrasound, ultrasound combined with liposome, ultrasound combined with microbubbles and liposome, and ultrasound combined with microbubbles respectively. The groups of negative control and positive control were treated by naked plasmid and liposome respectively, and then they were used to transfect plasmids pcDNA3. 1-EGFP/hsv_1tk. The transfection effieiencies were observed qualitatively by fluorescence microscope and quantitatively by flow cytometry ( FCM ) . The expression of hsv_1tk was detected by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: MTT assays showed that the transfection had no significant inhibition to cell viability under the conditions of the microbubble concentration ( 0.5 W/cm~2 ), frequency ( 1 MHz ) ,microbubble concentration (0.56 x 10~(11)/L), and the interval of every 8 s ultrasound exposure time ( 1s). Under the fluorescence microscope, the green fluorescence intensity for the group of ultrasound combined with microbubbles and liposome was the greatest.The analysis of FCM showed that the transfection efficiency in group of ultrasound and microbubbles was higher than that in group of ultrasound[ ( 11.74 ± 0. 19) % vs(2.19 ± 0.22 ) % ]. Furthermore, the transfection efficiency in group of ultrasound combined with microbubbles and liposome was ( 25.62 ± 0. 08 ) %, which was the highest among all groups ( P < 0.05 ). The detection of RT-PCR showed the expression of hsv_1 tk in group of ultrasound combined with microbubbles and liposome was the highest among the groups ( P < 0. 05 ). CONCLUSION : Under the conditions of the optimal ultrasound parameters, ultrasound- mediated microbubbles can not only independently promote the hsv_1 tk gene transfection, but also strengthen the transfection efficiency of liposome. Meanwhile it can also be effectively expressed in SKOV_3.
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