Research on the mechanism and security of MDR1 gene transfection into the bone marrow mononuclear cells of rabbit with adenovirus vector promoted by ultrasonic microbubble

Abstract

Objective To explore the mechanism and safety of miltidrug-resistance gene 1(MDR1)transfection into the bone marrow mononuclear cells of rabbit in vitro with the adenovirus vector promoted by ultrasonic microbubble.Methods Bone marrow mononuclear cells of rabbits were collected and divided into 5 groups after cultured in the 6-well plate according to the different experimental conditions(MDR1 gene was transferred into the cells with or without ultrasound irradiation and microbubbles)：conventional culture group (A)，Ad5-MDR1 group(B)，Ad5-MDR1+ultrasound irradiation group(C)，Ad5-MDR1+microbubbles group(D)，Ad5-MDR1+ultrasound irradiation+microbubbles group(E).The positive transfection rate of MDR1 gene in mononuclear cells of different groups were tested by flow cytometry,and the survival rate of cells in different periods were tested by trypan blue exclusion method.Moreover,the appearance and ultramicrostructure of cells were observed by electronmicroscope.Results ①The transfection rate of MDR1 gene in different groups were 0.39%±0.11%，5.03%±0.35%，4.93%±0.38%，5.25%±0.80%and 19.93%±1.51%respectively.The transfection rate of MDR1 gene in group E was higher than those in other groups(P＜0.05).②Compared with those in control groups(group A，B，C and D)，the transfection rate in group E was significant raised by ultrasound irradiation and microbubbles.However,there were no significant difference in survival rate of cells between the five groups(P＞0.05).③After ultrasonic irradiation，there were transient holes on the cell membrane，which could disappear after irradiation by ultrasound for 24 hours.And the temporary swelling of organelles was reversible.Conclusions Microbubbles irradiated by ultrasound can cause small transient holes on eell membrane and increase permeability of it，and enhance the transfection of MDR1 gene in bone marrow mononuclear cells withthe adenovirus vector，which is safe and available. Key words: Ultrasonography; Microbubbles; P-glycoprotein