[Inhibitory effect of human Col I A1 antisense oligodeoxynucleotide on collagen synthesis in hypertrophic scar fibroblasts].
OBJECTIVE
To investigate the inhibitory effect of Col I A1 antisense oligodeoxynucleotide (ASODN) transfection mediated by cationic liposome on Col I A1 expression in human hypertrophic scar fibroblasts.


METHODS
Scar tissue was obtained from volunteer donor. Human hypertrophic scar fibroblasts were cultured by tissue block method. The cells at passage 4 were seeded in a 6 well cell culture plate at 32.25 x 10(4) cells/well, and then divided into 4 groups: group A, liposome and Col I A1 ASODN; group B, Col I A1 ASODN; group C, liposome; group D, blank control. At 8 hours, 1, 2, 3 and 4 days after transfection, total RNA of the cells were extracted, the expression level of Col I A1 mRNA was detected by RT-PCR, the Col I A1 protein in ECM was extracted by pepsin-digestion method, its concentration was detected by ELISA method.


RESULTS
Agarose gel electrophoresis detection of amplified products showed clear bands without occurrence of indistinct band, obvious primer dimmer and tailing phenomenon. Relative expression level of Col I A1 mRNA: at 8 hours after transfection, group A was less than groups B, C and D (P < 0.05), and groups B and C were less than group D (P < 0.05), and no significant difference was evident between group B and group C (P > 0.05); at 1 day after transfection, groups A and B were less than groups C and D (P < 0.05), and there was no significant difference between group A and group B, and between group C and group D (P > 0.05 ); at 2 days after transfection, there were significant differences among four groups (P < 0.05); at 3 and 4 days after transfection, group A was less than groups B, C and D (P < 0.05), group B was less than groups C and D (P < 0.05), and no significant difference was evident between group C and group D (P > 0.05). Concentration of Col I protein: at 8 hours after transfection, group A was less than groups B, C and D (P < 0.05), groups B and C were less than group D (P < 0.05), and no significant difference was evident between group B and group C (P > 0.05); at 1 day after transfection, significant differences were evident among four groups (P < 0.05); at 2, 3 and 4 days after transfection, groups A and B were less than groups C and D (P < 0.05), and no significant difference was evident between group A and group B (P > 0.05).


CONCLUSIONS
Col I A1 ASODN can inhibit mRNA and protein expression level of Col I A1. Cationic liposome, as the carrier, can enhance the inhibition by facilitating the entry of ASODN into cells and introducing ASODN into cell nucleus.
Yuan
Jishan
J
Department of Orthopedics, the Affiliated People's Hospital of Jiangsu University, Zhenjiang Jiangsu, 212002, PR China. jishan0926@hotmail.com
Li
Tianzeng
T
Qi
Shaohai
S
D049109:Cell Proliferation; D002478:Cells, Cultured; D017439:Cicatrix, Hypertrophic / Q000378:metabolism*; D024042:Collagen Type I / Q000096:biosynthesis*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008081:Liposomes; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D011347:Procollagen / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection
D024042:Collagen Type I; D008081:Liposomes; D020319:Oligodeoxyribonucleotides, Antisense; D011347:Procollagen; D012333:RNA, Messenger