人Ⅰ型前胶原基因反义寡核苷酸抑制增生性瘢痕成纤维细胞胶原合成  

INHIBITORY EFFECT OF HUMAN COL Ⅰ A1 ANTISENSE OLIGODEOXYNEUCLEOTIDE ON COLLAGEN SYNTHESIS IN HYPERTROPHIC SCAR FIBROBLASTS

作　　者：袁即山[1] 利天增[2] 祁少海[2] 

机构地区：[1]江苏大学附属人民医院骨科,江苏镇江212002 [2]中山大学附属第一医院烧伤科

出　　处：《中国修复重建外科杂志》2009年第6期713-717,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：广东省重点科研项目资助(199827817);广东省医学科研联合攻关资助项目(98002)~~

摘　　要：目的探讨阳离子脂质体介导Ⅰ型前胶原基因(ColⅠA1)反义寡核苷酸(antisense oligodeoxyneucleotide,ASODN)转染对人增生性瘢痕成纤维细胞ColⅠA1表达的影响。方法取患者自愿捐赠瘢痕组织,采用组织块法培养人增生性瘢痕成纤维细胞并传代。于6孔板内按32.25×104个/孔的密度接种第4代细胞,根据转染液不同分为4组:A组为脂质体加ColⅠA1 ASODN,B组为ColⅠA1 ASODN,C组为脂质体,D组为空白对照。转染后8h,1、2、3、4d分别提取细胞总RNA,行RT-PCR后测定ColⅠA1 mRNA表达量;胃酶消化法提取ECM中ColⅠA1蛋白,ELISA测定其浓度。结果扩增产物经琼脂糖凝胶电泳检测显示条带清晰,无杂带、明显的引物二聚体及拖尾现象。ColⅠA1 mRNA相对表达量:转染后8h,A组小于B、C、D组,B、C组小于D组,比较差异均有统计学意义(P<0.05);B、C组间比较差异无统计学意义(P>0.05);转染后1d,A、B组小于C、D组(P<0.05),A、B组间和C、D组间比较差异无统计学意义(P>0.05);转染后2d,各组间比较差异均有统计学意义(P<0.05);转染后3、4d,A组小于B、C、D组,B组小于C、D组(P<0.05),C、D组间差异无统计学意义(P>0.05)。ColⅠ蛋白浓度:转染后8h,A组小于B、C、D组,B、C组小于D组,比较差异均有统计学意义(P<0.05),B、C组间差异无统计学意义(P>0.05);转染后1d,各组间比较差异均有统计学意义(P<0.05);转染后2、3、4d,A、B组小于C、D组,C组小于D组(P<0.05),A、B组间差异无统计学意义(P>0.05)。结论ColⅠA1 ASODN抑制ColⅠA1 mRNA和蛋白表达;阳离子脂质体作为载体有增强效果,促进ASODN进入细胞并在核内分布。Objective To investigate the inhibitory effect of Col I A1 antisense oligodeoxyneucleotide （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 32.25×10^4 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 diff

关 键 词：I型前胶原基因 反义寡核苷酸 增生性瘢痕 胶原 

分 类 号：R622[医药卫生—整形外科]