Transfection Of Human MxA Gene Into Mice Liver By Ultrasound Microbubble Destruction Technique

PARTⅠEnhanced gene expression of humanMxA gene in mice liver by ultrasound and lipid microbubble: eperimental studyObjective: To investigate whether local expression of exogenous gene could be enhanced by intravenous injection of gene and contrast agent with ultrasound wave irradiation in mice liver; to investigate biology safety of this technique.Method: 30 kunming mice were randomly divided into 5 groups with 6 mice in each group. Plasmid delivery group: 0.2ml normal saline solution containing 20μg PcDNA3.1-MxA was administrated by fast tail vein injection. Microbubble and plasmid group: 0.2ml contrast agent containing 20μg PcDNA3.1-MxA was administrated by fast tail vein injection. Ultrasound and plasmid group: 0.2ml normal saline solution containing 20μg PcDNA3.1-MxA was administrated by fast tail vein injection, and ultrasound with the frequency of 1MHz and intensity of 0.5W/cm2 was applied on the mice liver for 60s. Plasmid and ultrasound microbubble group: 0.2ml contrast agent containing 20μg PcDNA3.1-MxA was administrated by fast tail vein injection, and ultrasound with the frequency of 1MHz and intensity of 0.5W/cm2 was applied on the mice liver for 60s. After 7 days, the mice were killed by ceruical uertebra dislocation method. The lungs, hearts, spleens, kidneys, lungs, skeletal muscles, brains and bowels were harvested. Half quantitation of MxA protein were detected with immunofluorescence technique. The number of positive expression cells in each high power field(×400)were measured.Result: 1)Weakly positive expression of MxA gene were found in few liver cells in plasmid group; positive expression of MxA gene were found in few liver cells in microbubble and plasmid group; positive expression of MxA were found in a small number of liver cells in plasmid and ultrasound group and number of positive cells in each high power field was (15.17±2.31); positive expression of MxA were found in a great deal of liver cells in plasmid and ultrasound microbubble group and number of positive calls in each high power field was (92.83±4.45). There is significant difference between the above groups. 2)Positive expression of MxA were found in few spleen cells in plasmid and ultrasound microbubble group , while no positive call was found in lungs, hearts, spleens, kidneys,lungs, skeletal muscles, brains and bowels of rest groups. 3)Microscopic examination of liver HE staining in experimental group showed no difference with that of the controlling group.Conclusion: Local expression of exogenous gene could be significantly enhanced by intravenous injection of gene and contrast agent, with ultrasound wave irradiation in mice liver; the method may provide a safe and highly effective transfection method for genetic therapy of liver diseases.PARTⅡParameter of enhanced human MxA gene expression in mice liver by ultrasound and microbubble destruction techniqueObjective: To investigate effect of different ultrasound parameter on human MxA gene expression, by using ultrasound wave irradiation in mice liver with vein injection of target gene and contrast agent; to investigate human MxA gene expression in mice liver during different duration.Method: 1)Effect of different mechanical parameter on transfection efficiency: 20 kunming mice were randomly divided into 4 groups with 5 mice in each group. All the groups were treated with 0.2ml contrast agent containing 20μg MxA by fast tail vein injection, and ultrasound with the frequency of 1MHz and intensity of 0.25W/cm2, 0.5W/cm2,0.75W/cm2 and 1.0W/cm2 was applied on the mice liver for 60s. The mice were killed after 7days. The livers were harvested, and half quantitation of MxA protein were detected with immunofluorescence technique. 2) time-dependent human MxA expression in mice liver: 28 kunming mice were randomly divided into 7 groups with 4 mice in each group. All the mice were treated with 0.2ml contrast agent containing 20μg MxA by fast tail vein injection, and ultrasound with the frequency of 1MHz and intensity of 0.5W/cm2 was applied on the mice liver for 60s. all the groups were killed after 1,2,4,7,10,12,14 days, respectively. The livers were harvested, and half quantitation of MxA protein were detected with immunofluorescence technique.Result: number of positive expression liver cells was highest in 0.25W/cm2group, followed by 0.75W/cm2group (6 5.40±3.05)/HP and 1.0W/cm2group (5 0.40±2.07)/HP, number of positive expression liver cells was the lowest in 0.25W/cm2 group (30.00±2.55)/HP. MxA positive expression cells were found in 4 days after gene transfection, MxA expression was the highest in 7 days after transfection and positive cells could hardly be detected in 14 days.Conclusion: In a certain range, when frequency of ultrasound wave irradiation remained as unchanged, expression of human Maxi gene in mice liver can be enhanced with increased intensity of sound; transfection efficiency was the highest with intensity of 0.5W/cm2; MxA positive expression cells were found in 4 days after gene transfection, MxA expression was the highest in 7 days after transfection and positive cells could hardly be detected in 14 days with ultrasound and microbubble destruction technique.