摘要
目的:构建特异性shRNA腺病毒载体,使之干扰神经轴突过度生长因子(PTN)在胰腺癌细胞中的表达,探讨其对SD大鼠背根神经节神经元(DRGn)神经轴突的影响。方法:设计并合成4对靶向PTN单链寡核苷酸(ss oligo),经变性退火为双链寡核苷酸(ds oligo)插入穿梭质粒pENTR/U6载体,测序正确后经脂质体介导转染胰腺癌细胞,通过RT-PCR方法筛选对PTN沉默效果最佳的穿梭质粒pENTR/U6-shRNA,然后与腺病毒骨架质粒进行同源重组,筛选出正确重组子,用HEK293A细胞包装出表达shRNA-PTN的重组腺病毒并测定其病毒滴度,通过Western blot方法对腺病毒沉默PTN效果予以检测。shRNA-PTN腺病毒构建成功后感染胰腺癌PC-2细胞,然后和DRGn共培养,观察DRGn生长分化和形态特征。结果:测序结果提示所构建的pENTR/U6-shRNA质粒正确,并从4对ds oligos筛选出对PTN沉默效果最佳的穿梭质粒pENTR/U6-shRNA,经同源重组后成功构建了介导shRNA-PTN复制缺陷型重组腺病毒。转染胰腺癌细胞后,Western blot检测显示shRNA-PTN腺病毒感染胰腺癌细胞后对PTN蛋白的沉默效果7 d后达最佳效果。感染shRNA-PTN腺病毒的胰腺癌PC-2细胞和DRGn共培养,DRGn神经轴突的生长和延长受抑制,以第7天最为明显。结论:成功地构建介导shRNA-PTN复制缺陷型重组腺病毒;感染shRNA-PTN腺病毒的胰腺癌PC-2细胞和SD鼠DRGn共培养可以抑制DRGn神经轴突的生长。
AIM： To construct the replication-incompetent recombinant adenovirus mediated shRNA to inhibit the neurite growth-promoting factor （ Pleiotrophin, PTN） in pancreatic carcinoma and to study the inhibitory effect of shRNA-PTN recombinant adenoviruses on the neurite＇s growth of dorsal root ganglion neurons （DRGn）. METHODS： Four pairs of complementary single-stranded oligonucleotides （ss oligo） were designed and synthesized and then they were annealed to create a double-stranded oligonucleoUde （ ds oligo）. The ds oligos were cloned into pENTR/U6 vector to produce the shuttle plasmid pENTR/U6-shRNA, which was transduced into pancreatic carcinoma cells by liposome after sequencing. The plasmid with good silence effect was chosen by RT-PCR to perform the LR recombination reaction to the adenovirus backbone plasmid. The expression clone was transfected into HEK293A cell to produce adenovirus. The silence of the recombinant adenovirus against PTN was detected by Western blot. After DRGn was co-cultured with pc-2 cell infected by shRNA-PTN recombinant adenovirus, the morphological changes of DRGn were observed. RESULTS： The pENTR/U6-shRNA shuttle plasmid was constructed and confirmed by sequencing. The recombinant adenovirus mediated shRNA against PTN was constructed. The best silence effect of the adenovirus against PTN was detected by Western blot on the 7th day after the pancreatic carcinoma cell was transfected. The growth of DRGn neurites was inhibited after the co-culture of DRGn with pc-2 cell which was infected by shRNA-PTN recombinant adenovirus. CONCLUSION： The PTN SiRNA recombinant adenovirus has been constructed and the silence effect against PTN in pancreatic carcinoma cell has been confirmed. The growth of DRGn neurites can be inhibited by shRNA-PTN recombinant adenovirus.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2007年第9期797-800,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
陕西省科技攻关项目资助(2007K09-04)
