Experiment of ultrasound microbubble mediating gene EGFP transfecting retinal ganglial cells in vivo

Abstract

Objective To investigate whether ultrasound microbubble could mediate gene EGFP transfecting retinal ganglial cells (RGCs) in vivo,whether this transfection way is more effective than the orthodox way and whether this way could cause damage of RGCs.Methods Fifty SD rats were randomly divided into 4 groups:the normal control group (n =5),plasmid group (n =15),plasmid + ultrasound group (n =15)and ultrasound microbubble group (n =15).The normal control group was injected 5 μL normal saline into vitreous cavity.The plasmid group was injected 5 μL plasmid.The plasmic + ultrasound group was injected 5 μL plasmid,then we exposed rats eyeballs to 0.5 W/cm2 ultrasonic wave immediately for 60 s( the exposure time accounting for 1/3:the exposure time is 5 s,then pause 10 s,total time 60 s.The ultrasound microbubble group was injected 5 μL suspension of plasmid and microvesicle,then we exposed rats eyeballs as the above way immediately.Seven days later,we made stretched preparation and longitudial frozen section of retina,and RT-PCR of EGFP mRNA of retina.Then fluorescence microscope was used to observe stretched preparation and EGFP expression in RGCs.We counted the number of RGCs to observe the damage situation.RGCs EGFP mRNA was detected through RT-PCR semi-quantitatively.Results The efficiency of ultrasound microbubble mediating gene EGFP transfecting RGCs was significantly higher than the normal control group,plasmid group and plasmic + ultrasound group,and this transfection way didn' t cause damage of RGCs.Conclusion By exposing eyeball to ultrasound of low frequency transduction,ultrasound can mediate EGFP gene transfecting RGCs safely and effectively. Key words: Ultrasonography; Retinal ganglial cells; Gene therapy; Enhanced green fluorescent protein; Gene transfection