【摘要】 目的:探讨丝裂素活化蛋白激酶(MAPK)反义寡核苷酸(ODN)对血管紧张素Ⅱ(Ang Ⅱ)诱导的心肌成纤维细胞c-myc基因及其细胞增殖的抑制效应。方法:MAPK反义ODN转染培养新生大鼠心肌成纤维细胞,Western Blot法结合p-81滤纸法测定MAPK活性;Northern Blot法检测c-myc mRNA的表达;[~3H]TdR掺入和[~3H]脯氨酸接入测定细胞DNA和胶原蛋白的合成。结果:MAPK反义ODN显著抑制Ang Ⅱ诱导的MAPK蛋白表达及其活性;显著抑制c-myc基因的表达以及细胞DNA和胶原蛋白的合成。结论:MAPK反义ODN特异性下调MAPK的活性,有效抑制了Ang Ⅱ诱导的c-myc基因的表达以及心肌成纤维细胞的增殖和胶原蛋白的合成。更多还原

【Abstract】 AIM: To investigate the inhibitory effect of down-regulating mitogen activated protein kinase (MAPK) on c-myc gene expression and further on cardiac flbroblast proliferation. METHODS: Cultured neonatal rat cardiac fibroblasts was pretreated with a phosphoro-thioate-protected 17-mer antisense MAPK oligodeo-xynucleotide (ODN) directed against the initiation of translation sites of the p42 and p44 MAPK isoforms by liposomal transfection. A 17-mer sense and mismatch sequence MAPK ODN were used as controls. After liposomal transfecting, cells were exposed to angiotensin Ⅱ (Ang Ⅱ ) 10 nmol·L-1 for 5 min and then harvested in lysis buffer. MAPK activity was measured by Western blot and P-81 phosphocellulose filter paper method by using [γ-32P] ATP and myelin basic protein as substrates, c-myc mRNA expression stimulated by Ang Ⅱ for 30 min was measured by Northern blot. DNA synthesis and collagen protein synthesis induced by Ang Ⅱ for 24 h were measured by [3H]thymidine incorporation and [3H]Proline incorporation, respectively. RESULTS: antisense ODN 0.2 μmol·L-1 reduced Ang Ⅱ -induced MAPK activities by 72 %, MAPK protein expression by 80 %, and suppressed c-myc mRNA expression by 97 % , respectively. [3H]thymidine incorporation and [3H]proline incorporation in Ang Ⅱ -induced cardiacflbroblast were inhibited by 59 % and 58 %, respectively. CONCLUSION: A 17-mer MAPK antisense oligonucleotide directed againsts the initiation of translation sites of MAPK could specifically inhibit Ang Ⅱ -stimulated cultured neonatal rat cardiac flbroblast proliferation through down-regulating MAPK activity and further depleting c- myc mRNA expression.更多还原