目的构建RNA干扰(RNAi)重组体抑制黏着斑激酶(FAK)表达,并探讨其对结肠癌多细胞球体(MCSs)形成的影响。方法针对FAK cDNA序列设计并合成1对特异性的含有短发卡的寡核苷酸序列及其对照序列,经退火后插入pGenesil-1中构建重组体。经双酶切鉴定和DNA测序后,用脂质体2000将其转染结肠癌HT29细胞,并用G418稳定筛选,采用Real-time PCR和Western blot检测干扰前后FAK在HT29内的表达变化,并作细胞悬浮培养观测靶向干扰FAK对MCSs形成的影响。结果双酶切和测序鉴定证实插入序列完全正确;靶向干扰FAK后,其mRNA和蛋白表达分别显著下调(79.20±2.97)%和(78.47±4.39)%,且细胞不易聚集形成MCSs。结论成功构建FAK靶向RNAi重组载体,显著地抑制FAK表达,并能有效地抑制MCSs的形成。 Objective To construct RNA interference (RNAi) recombinant plasmids to inhibit the expression of focal adhesion kinase (FAK) and to investigate its effect on the formation of colon cancer multicellular spheroids (MCSs). Methods A pair of specific short hairpin-containing oligonucleotide sequences and their control sequences were designed and synthesized based on the FAK cDNA sequence. After annealing, the recombinant plasmid was inserted into pGenesil-1. After double enzyme digestion and DNA sequencing, the recombinant plasmid was transfected into HT29 cells with lipofectamine 2000 and stably transfected into HT29 cells with G418. Real-time PCR and Western blot were used to detect the expression of FAK in HT29 cells before and after interference Effect of Targeted Interference FAK on MCSs Formation in Suspension Cultures. Results The results of double enzyme digestion and sequencing confirmed that the inserted sequence was correct. The mRNA and protein expression of FAK were significantly down-regulated (79.20 ± 2.97)% and (78.47 ± 4.39)%, respectively, and the cells were not easily aggregated to form MCSs. Conclusion FAK targeting RNAi recombinant vector was successfully constructed, which markedly inhibited the expression of FAK and effectively inhibited the formation of MCSs.