Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery · 2009
[Inhibitory effect of human Col I A1 antisense oligodeoxynucleotide on collagen synthesis in hypertrophic scar fibroblasts].
Аннотация на английскомСтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод пока не подготовлен
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Yuan Jishan, Li Tianzeng, Qi Shaohai
Аннотация
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).
CONCLUSION: 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.
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).
CONCLUSION: 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.