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用前期设计并合成好的重组腺病毒质粒pAdsiALK2,经Pac I酶切后转染至HEK293细胞进行包装,制备重组腺病毒,扩增后进行病毒滴度测定,以重组腺病毒siALK2(AdsiALK2)感染MDA-MB-231细胞,以含RFP的腺病毒作为感染对照,并设空白对照组。通过RT-PCR验证MDA-MB-231细胞中ALK2表达显著下降;MTT法、平板集落形成试验、细胞划痕试验及Transwell侵袭试验证实MDA-MB-231/siALK2组相比MDA-MB-231/RFP组细胞的增殖活力、集落形成率及划痕愈合率均显著降低(P<0.05),穿膜细胞数也明显减少(P<0.05),而MDA-MB-231/RFP组与空白对照组比较,差异无统计学意义(P>0.05)。该研究表明,下调ALK2表达后可以在体外抑制乳腺癌MDA-MB-231细胞的增殖、迁移与侵袭。
Recombinant adenovirus plasmid pAdsiALK2 was designed and synthesized in advance and was digested with Pac I and then transfected into HEK293 cells for packaging. The recombinant adenovirus was prepared and amplified for viral titers. The recombinant adenovirus was then infected with the recombinant adenovirus siALK2 (AdsiALK2) MDA-MB-231 cells with RFP-containing adenovirus as an infection control, and set the blank control group. The expression of ALK2 in MDA-MB-231 cells was significantly lower than that of MDA-MB-231 / siALK2 group by MTT assay, The cell viability, colony formation rate and wound healing rate of RFP group were significantly decreased (P <0.05), and the number of transmembrane cells was also significantly decreased (P <0.05), while MDA-MB-231 / RFP group and blank control group The difference was not statistically significant (P> 0.05). The study shows that down-regulation of ALK2 expression can inhibit the proliferation, migration and invasion of breast cancer MDA-MB-231 cells in vitro.