Parameters optimization of ultrasound mediated gene transfection in HepG2 / 中华超声影像学杂志

Objective To investigate the feasibility of transfecting gene into HepG2 by therapeutic ultrasound combined with microbubble,and to explore the optimal ultrasonic parameters for high transfection efficiency.Methods HepG2 cells were cross-interval planted in 24-well plates.EGFP plasmid DNA and microbubbles were added to the cultured HepG2 and three parameters of the therapeutic ultrasound were optimized.Firstly,set ultrasonic intensity gradient (group A,from A1 to A5),which were 0.4 W/cm,0.8 W/cm2,1.2 W/cm2,1.6 W/cm2 and 2.0 W/cm2 respectively.Secondly,set the duty cycle gradient group (group B,from B1 to B3),which were 10％,20％ and 50％ respectively.Lastly,set the exposure time group(group C,from C1 to C3),which were 30 s,90 s and 180 s.After 24 hours,the expression of GFP was observed by fluorescence microscopy,the transfection rates was detected by flow cytometry,the cell death were assessed by trypan blue exclusion.Results The plasmid transfection efficiency was varied under different ultrasonic parameters.Within a certain range,the increasing of parameters can improve the transfection efficiency,but when the parameters were too large (eg.the intensity＞1.6 W/cm2 or 50％ duty cycle),the actual transfection efficiency decreased due to the increased cell death.When the ultrasonic intensity was 1.2 W/cm2,the transfection efficiency and mortality were better than the other subgroups of group A.When the duty cycle was 20％,the transfection efficiency and mortality rate were better than the other subgroups of group B.Continue to increase the exposure time over 90 s was not statistically significant (P ＞0.05) for transfection efficiency and cell mortality.Conclusions The ultrasound-targeted microbubble destruction is an efficient method for gene delivery in vitro.The transfection efficiency vary greatly under different parameters,so optimization parameters is conducive to the promotion of gene transfection.

Changes of hepatocellular carcinoma cell structures during apoptosis induced by Survivin antisense oligonucleotide / 中国普通外科杂志

ObjectivesTo investigate the changes of hepatocellular carcinoma cell line SMMC-7721 in cell ultrastructure and skeleton during apoptosis induced by Survivin ASODN.MethodsSurvivin ASODN was transfected into SMMC-7721 cells mediated by DOTAP liposomal reagent. The changes of Survivin protein and mRNA expression after transfection were assessed by Western blot and in situ hybridization. The apoptotic rate was detected by flow cytometer. Cell ultrastructure and skeleton were observed by electron microscope and confocal microscope. Caspase-3 was assessed by kinase activity assess method. Results The expression of Survivin protein and mRNA decreased from 69.59 and 75.6 to 10.71 and 22.9 respectively. The cell ultrastructure showed apoptotic changes. Cell skeleton microfilament system changed after Survivin ASODN transfection. The average intensity of microfilament system fluorescent decreased from 190 to 64. The activity of Caspase-3 increased from 0.015 3 to 0.099 2.ConclusionsTransfection of ASODN targeted to Survivin mRNA by DOTAP liposomal transfection reagent down-regulated the expression of Survivin protein and mRNA in SMMC 7721 cell line and activate Caspase-3, which in turns induced cell apoptosis.