The Experimental Study Of The Influence On PEX Gene Transfection On Rat Glioma C6 Cells By Ultrasound-targeted Microbubble Destruction

Objectives(1)To investigate the feasibility of pEGFP-C1-PEX transfection into rat glioma C6 cells,and observe the influence of PEX gene on C6 cells,which was mediated by SonoVue combined with ultrasonic irradiation.(2)To explore the feasibility and advantages of liposome transfection of PEX gene to rat glioma C6 cells enhanced by ultrasound-targeted microbubble destruction(UTMD),jointing UTMD and liposome for PEX gene in rat glioma C6 cells transfection.Methods(1)The growing-well rat glioma C6 cells were cultured in vitro and divided into6 groups,control group,ultrasound+ microbubbles group,plasmid+ microbubbles group,plasmid+ ultrasound group,plasmid+ ultrasound+ microbubbles group.Ultrasonic irradiation parameters set as 1MHz for the frequency,1.0W/cm2 for irradiation power,duration for 30 sec,duty cycle for 20%.The expression of enhanced green fluorescent protein(EGFP)was observed by fluorescence microscopy,the percentage of fluorescent cells in each group was measured by flow cytometry so asto evaluate the transfection efficiency,the expression of PEX mRNA was detected by real-time polymerase chain reaction(RT-PCR),and the flow cytometry was used to analyze the cell cycle distribution after each group were treated accordingly for twenty-four hours.(2)The rat glioma C6 cells cultured in vitro were divided into control group,microbubble group,ultrasound group,ultrasound+ microbubble group,liposome group,ultrasound+ microbubble+ liposome group.Twenty-four hours after appropriate treatment,the expression of EGFP was observed by the fluorescence microscope,and the transfection efficiency was detected using the flow cytometry.Results(1)Fluorescence microscopy showed the largest number of green fluorescent cells,flow cytometry performed the highest transfection efficiency,the RT-PCR showed specific expression of PEX electrophoresis band,and the flow cytometry analysis showed that the percentage of cell cycle distribution G0/G1 phase was significantly increased in plasmid+ ultrasound+ microbubbles group,compared with other groups,the differences were statistically significant(all P<0.05).(2)Under fluorescence microscopy,the expression of green fluorescent protein in ultrasound+ microbubbles+ liposome group was numerous,significantly more than the other groups.The flow cytometry was used to detect the transfection rate,showing that ultrasound+microbubble group was(8.59±1.94)%,the liposome group was(13.71±2.99)%,comparing the two groups,the latter was significantly higher than the former,as well as the difference was statistically significant(P<0.05);Then the ultrasound+ microbubble+ liposome group was(18.31±2.66)%,which was significantly higher than other groups,the differences were statistically significant(all P<0.05).Conclusions(1)Sonove combined with ultrasound irradiation could enhance the transfection of PEX gene in rat glioma C6 cells.PEX gene can affect the cell cycle to inhibit theproliferation of rat glioma C6 cells in vitro experiments.(2)UTMD can enhance the transfection of liposome-mediated PEX gene in rat glioma C6 cells.