反义c-mycRNA的表达对大鼠血管平滑肌细胞c-mycmRNA和c-Myc蛋白水平的影响

为探讨反义ｃ－ｍｙｃ逆转录病毒载体是否影响血管平滑肌细胞表达ｃ－ｍｙｃ ｍＲＮＡ和ｃ－Ｍｙｃ蛋白水平，用基因重组方法，将第二外显子及其两则部分内含子共１．５３ｋｂ序列的ｃ－ｍｙｃ基因片段反向连接于Ｐ＾ＬＸＳＮ的５′长末端重复序列下游，通过Ｌｉｐｏｆｅｃｔｉｎ将反义ｃ－ｍｙｃ逆转录病毒导入ＰＡ３１７包装细胞系，并经Ｇ４１８筛选获得抗性克隆。反义－ｃ－ｍｙｃ逆转录病毒上清液转染的平滑肌细胞经ＤＮＡ印迹

Effect of Antisense c-myc RNA On c-mycmRNA and c-Myc Protein Expression inRat Aortic Smooth Muscle Cells

Aim Antisense c-myc retrovirus vector was con-structed and was introduced into smooth muscle cell(SMC) by Lipofection. The effect of antisence c-mycRNA on c-myc mRNA and c-myc protein expression inSMC was investigated , exploring the new strategy forgene therapy for diseases of c-myc overexpression.Methods 1. 53 kb c-myc fragment including a partof the first intron, the second exon and first 8 bp ofthe second intron was placed under 5''LTR downstreamof in inverted orientation. Antisense c-myc retrovirus vector was introduced into PA317 packagingcells by Lipofection and selected by G418. SMC wereinfected with this recombinant retrovirus. Northernblot, and Western blot hybridizations were performedin SMC transfected by antisense c-myc retrovirus vec-tor to observe whether it was integrated into SMC ge-nomic DNA, whether antisense c-mycRNA expressedin SMC and whether the expression of antisense c-mycRNA reduced c-myc mRNA expressed in SMC andwhether the expression of antisense c-myc RNA re-duced c-myc mRNA and c-myc protein levels.Results Southern blot analysis confirmedthe inte-gration of the antisense c-myc retrovirus vector intoSMC genomic DNA. Northern blot analysis demon-strated the expression of antisense c-myc RNA and thelatter recuced the level of c-myc mRNA. Westernblot analysis showed antisense c-myc RNA inhibitedthe translation of c-myc protein, whereas sense c-mycRNA didn''t have such effect.Conclusion Antisense c-myc RNA inhibited the ex-pression of c-myc mRNA and c-myc protein in a se-quence-specific manner. Antisense c-myc RNA is anefficient tool to inhibit the expression of c-myc gene ex-pression. This investigation provides a basis for fu-ture study using antisense c-myc RNA for gene therapyfor diseases of c-myc overexpression inducing malignanttumors and some cardiovascular diseases.