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目的构建含有白细胞介素8(IL-8)基因的重组腺病毒,并观察其对BT549乳腺癌细胞增殖、细胞周期以及迁移能力的影响。方法以143B细胞c DNA为模版,PCR扩增IL-8基因,与穿梭质粒p Ad Track-TO4连接,构建重组穿梭质粒p Ad Track-TO4-IL-8,由PmeⅠ线性化并电转入Ad Easier细菌中,获得重组腺病毒质粒p Ad IL-8,PacⅠ酶切经LipofectamineTM2000介导转入HEK293细胞进行包装扩增,检测滴度;反转录PCR检测BT549细胞中IL-8 mRNA的水平,ELISA检测BT549细胞培养上清液中IL-8的水平;流式细胞术检测细胞周期情况;MTT法检测细胞增殖能力;划痕愈合实验检测细胞迁移能力。结果PCR以及测序证实p Ad Track-TO4-IL-8构建成功;p Ad IL-8经酶切证实重组正确;重组腺病毒Ad IL-8感染BT549细胞,可高表达IL-8。过表达IL-8的BT549细胞迁移能力增强,但其增殖活性无明显变化、将细胞周期阻滞于S期。结论过表达IL-8可促进BT549细胞的迁移。
Objective To construct a recombinant adenovirus containing interleukin 8 (IL-8) gene and observe its effect on the proliferation, cell cycle and migration of BT549 breast cancer cells. Methods The IL-8 gene was amplified by PCR using c DNA of 143B as a template. The recombinant plasmid pAdTrack-TO4-IL-8 was constructed by ligating with shuttle plasmid pAdTrack-TO4. Easier bacterium, the recombinant adenoviral plasmid pAd IL-8 was obtained. PacI digested by LipofectamineTM2000 was transfected into HEK293 cells for packaging and amplification, and the titer was detected. The level of IL-8 mRNA in BT549 cells was detected by reverse transcription polymerase chain reaction (RT-PCR) The level of IL-8 in the culture supernatant of BT549 cells was detected by ELISA. The cell cycle was detected by flow cytometry. The cell proliferation was measured by MTT assay. The wound healing assay was used to detect the cell migration. Results PCR and DNA sequencing confirmed that pAd-Track-TO4-IL-8 was successfully constructed. Recombinant pAd-8 was verified by restriction enzyme digestion. The recombinant adenovirus Ad-IL-8 was highly expressed in BT549 cells. BT549 cells overexpressing IL-8 enhanced their migration ability, but their proliferative activity did not change significantly, blocking the cell cycle in S phase. Conclusion Overexpression of IL-8 can promote the migration of BT549 cells.