Transfection optimization of α_1(I) procollagen gene antisense oligodeoxynucleotide to human hypertrophic scar fibroblasts

Objective To optimize the transfection of α_1(I) procollagen gene antisense oligodeoxynucleotide to human hypertrophic scar fibroblasts.Methods Passage 2 to passage 6 fibroblasts grown out of human hypertrophic scars were subcultured in the 6-well plates in different cell counts ranging from 14.25× 104/well to 39.00× 104/well.The sequence-known antisense oligonucleotide was labeled at the 5'-end with FITC when synthesizing and phosphothioating,and the ASODNs were transfected with cationic liposome Oligofectamine TM to these cells.Then the locations of fluorescence in the cells were observed under laser scanning confocal microscopy 24 h after transfection.At the same time,the percentage of ASODN transfected cells and apoptic cells were counted by flow cytometry.The factors affecting the transfection were optimized according to the results of flow cytometry.Firstly,the cell counts were optimized with 2 μl of Oligofectamine TM and 200 nmol/L of ASODN;Secondly,the concentration of ASODN was optimized with the optimal cell count and 2 μl of Oligofectamine TM ;Lastly,the best volume of cationic liposome was determined by applying the chosen optimal cell count and ASODN concentration.Results (1) No apoptosis occurred in this experiment.After transfection the ASODNs located both in the cytoplasm and the nuclei.(2) The percentage of fluorescent cells was 74.0% when the inoculated cells was 14.25×104/well,and 73.3% to 30.00× 104/well, 94.3% to 32.25× 104/well, 64.2% to 39.00× 104/well,respectively.(3) The percentage of fluorescent cells was 38.1 % when the transfecting concentration of ASODN was 100 nmol/L,and 62.6 % to 150 nmol/L, 49.5 % to 200 nmol/L,and 35.8 % to 250 nmol/L,respetively.(4)The percentage of fluorescent cells was 77.01 % when 2 μl of liposome was used during transfection, 85.05 % when 3 μl, 71.64 % when 4 μl,respectively.ConclusionWithin the 6-well plate,the optimal transfection condistions of ASODN to the human hypertrophic scar fibroblasts were as follows:the cell count of 32.25× 104/well,concentration of ASODN 150 nmol/L,and cationic liposome Oligofectamine TM ,2 μl,respectively.Transfection efficiency could be improved after transfection optimization,and this would help set foundation for further investigation.