Yao Jun, Ma Qing-yong, Wang Lian-cai, Zhang Min, Shen Su-gang
Department of Hepatobiliary, the First Hospital of Xi'an Jiaotong University, Xi'an 710061, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Sep;23(9):797-800.
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).
Four pairs of complementary single-stranded oligonucleotides (ss oligo) were designed and synthesized and then they were annealed to create a double-stranded oligonucleotide (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.
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.
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.
构建无复制能力的重组腺病毒介导的短发夹RNA（shRNA），以抑制胰腺癌中促神经突生长因子（多效生长因子，PTN）的表达，并研究shRNA-PTN重组腺病毒对背根神经节神经元（DRGn）神经突生长的抑制作用。
设计并合成四对互补单链寡核苷酸（ss oligo），然后退火形成双链寡核苷酸（ds oligo）。将ds oligo克隆到pENTR/U6载体中以产生穿梭质粒pENTR/U6-shRNA，测序后通过脂质体转染到胰腺癌细胞中。通过逆转录聚合酶链反应（RT-PCR）选择沉默效果良好的质粒，与腺病毒骨架质粒进行LR重组反应。将表达克隆转染到HEK293A细胞中以产生腺病毒。通过蛋白质免疫印迹法检测重组腺病毒对PTN的沉默作用。将DRGn与感染了shRNA-PTN重组腺病毒的pc-2细胞共培养后，观察DRGn的形态变化。
成功构建了pENTR/U6-shRNA穿梭质粒并经测序确认。构建了重组腺病毒介导的针对PTN的shRNA。在胰腺癌细胞转染后第7天，通过蛋白质免疫印迹法检测到腺病毒对PTN的最佳沉默效果。DRGn与感染了shRNA-PTN重组腺病毒的pc-2细胞共培养后，DRGn神经突的生长受到抑制。
已构建PTN小干扰RNA（SiRNA）重组腺病毒，并证实其对胰腺癌细胞中PTN的沉默作用。shRNA-PTN重组腺病毒可抑制DRGn神经突的生长。
