摘要
构建一个含1．53kb反义c－myc片段的逆转录病毒载体PAS-c-myc，将其通过Lipofectin导入PA317细胞并经G418筛选获得分泌病毒上清液的阳性克隆。病毒上清波感染血管平滑肌细胞（SMC）经G418筛选获得抗性克区。DNA印迹杂交证实了反义c-myc片段整合于SMC基因组中。RNA印迹杂交证实了反义c－mycRNA在SMC中得到了表达，且显著降低了SMC中c－mycmRNA的水平。蛋白印迹杂交证实了反义c－mycRNA抑制了c－myc蛋白的翻译。提示：c－myc基因表达在SMC增殖中起重要作用。
c-myc gene expression plays a pivotal role in cell proliferation. Antisense RNA mayspecifically abolish the function of target gene. A retroviral vector,pAS-c-myc was constructed. 1. 53kbc-myc fragment including part of the lst intron,2nd exon and the lst 8 bps of 2nd intron was placedunder 5' LTR of pLXsN in inverted orientation. PAS-c-myc was introduced into PA317 packaging cells bylipofectin. G418-resistant clones were isolated. SMCs were infected with this recombinant virus andselected by G418. Southern blot analysis confirmed the insertion on antisense c-myc fragment intothe genomic DNA of SMC. Antisense c-myc RNA expression was observed and it reduced significantly the level of c-myc mRNA in SMCAS-c-myc by Northern blot analysis. Western blot analysisshowed antisense c-myc RNA significantly inhibited c-Myc protein transistion. Antisense c-mycRNA really suppressed SMC proliferation and its 3H-TdR incorporation. Conclusions: (1 ) Theseresults suggest antisense c-myc mRNA significantly diminish the levels of c-myc mRNA and c-Mycprotein ; (2) c-myc expression plays an important part in SMC proliferation; (3) This investigationprovides a basis for future studies designed to treat diseases with c-myc overexpression using antisense c-myc RNA strategy.
出处
《临床心血管病杂志》
CAS
CSCD
北大核心
1998年第4期235-239,共5页
Journal of Clinical Cardiology
