中文摘要：

目的为探讨S1P2受体介导血管内皮细胞衰老的机制，构建和筛选特异性S1P2受体shRNA腺病毒载体。方法设计并合成4对靶向S1P2受体的单链寡核苷酸（ss oligo），经变性退火为双链寡核苷酸（ds oligo）插入穿梭质粒pRNAT-H1.1／Shuttle载体，测序正确后经脂质体介导转染人肺动脉内皮细胞（HPAEC），通过RT-PCR方法筛选对S1P2受体基因沉默效果最佳的穿梭质粒pRNAT-H1．1／Shuttle-shRNA，提取质粒DNA，经双酶切，回收S1P2受体基因沉默效果最佳的shRNA片段，亚克隆到腺病毒表达载体（pAdeno-X），筛选出正确重组子，转染HEK293A细胞，收集重组的腺病毒，测定病毒滴度，通过Western blot方法检测s1P2受体沉默效果。结果测序结果证实所构建的pRNAT-111．1／Shuttle-shRNA质粒正确，并从4对ds oligos筛选出对S1P2受体沉默效果最佳的穿梭质粒pRNAT-H1.1/Shuttle-shRNA，经亚克隆到腺病毒表达载体。转染人肺动脉内皮细胞后，Western印迹检测显示构建的腺病毒载体pAdeno-shRNA对S1P2受体蛋白的沉默有明显效果。结论成功地构建和筛选出介导特异性shRNA-S1P2的重组腺病毒。

英文摘要：

Objective To construct and screen the recombinant adenovirus vector with S1P2 receptor-targeted shRNA for studing the mechanisms of S1P2 receptor-mediated aging of vascular endothelial cells （ECs）. Methods 4 pairs of complementary single stranded oligo-nucleotides （ss oligo） were designed and synthesized and then were annealed to create a double stranded oligonucleotide （ds oligo）. The ds oligos were cloned into pRNAT-H1.1/Shuttle vector and transduced into human pulmonary artery endothelial cells （HPAECs） after checking the correct sequence. By RT-PCR the shuttle plasmids pRNAT-H1. 1/Shuttle-shRNA were acquired, extracted DNA plasmids then cut by double-enzyme and retrieved the segment with the best silence effect on S1P2 receptor-sbRNA. Then the obtained recombinant plasmids were transformed into adenovirus vector-pAdeno-X to screen the correct recombinant plasmids, be transformed into HEK293 cells to measure the titre of adenovirus. The silence effect of S1P2 were detected by Western blot. Results The recombinant plasmids pRNAT-H1.1/Shuttle-shRNA were constructed and confirmed by sequencing, and the best recombinant plasmids with silence effect against S1P2 receptor was screened and was sub-cloned into adenovirus vector. The recombinant adenovirus mediated shRNA against S1P2 was constructed and transfected to HPAEC. The significant silence of S1P2 expression was identified by Western blot after transfected HPAECs. Conclusions The recombinant adenovirus vector with S1P2-targeted shRNAs is successfully constructed and screened.