靶向乳腺癌干细胞树突细胞瘤苗核酸抗原的制备

摘要

The study was to enrich MCF 7 breast cancer stem cells by mammosphere culture using serum free mediumand prepare mRNA nucleic acid antigen for preparation of dendritic cell vaccine targeting on breast cancer stem cell. MCF 7 breast cancer stem cell was cultured and enriched by using serum free medium, and confirmed by identification of cellular sur face marks by using flow cytometer, clone forming capability test by agar assay, and tumorigenic ability test in vivo by using NOD SCID mouse. The mRNA was amplified by using T7 mMESSAGE mMACHINE kit with template of total RNA extracted from MCF 7 breast cancer stem cells. The result showed that MCF 7 breast cancer cell grouped and formed mammosphere in cultural suspension by using serum free media. About 90. 16% cells within mammosphere possessed CD+44/CD-24 phenotype. The ability of clone forming and capability of tumor initiating in NOD/SCID mouse with low dose were stronger in suspension cells than that in adhesive cells. The mRNA molecule amplified in vitro could be as specific nucleic acid antigen of breast cancer stem cells. We proposed that the high percent of MCF 7 breast cancer stem cells could be obtained by mammosphere culture u sing serum free medium, and mRNA amplified in vitro could be as specific nucleic acid antigen of breast cancer stem cells. It lay a foundation for preparation of dendritic cell vaccine targeting on breast cancer stem cell.%目的 应用无血清培养基细胞球培养法富集MCF-7乳腺癌千细胞,制备乳腺癌干细胞mRNA核酸抗原,为制备靶向乳腺癌干细胞树突细胞瘤苗奠定基础.方法 应用无血清培养基悬浮培养法富集MCF-7乳腺癌细胞,经单克隆形成、表面标志检测、NOD-SCID小鼠成瘤等实验对其肿瘤干细胞特性进行鉴定后,利用T7 mMESSAGE mMACHINE试剂盒进行mRNA体外扩增.结果 乳腺癌细胞MCF-7在无血清培养基中可形成悬浮状细胞球,其中具有CD44+CD24-表面标志的细胞约占90.16%.该细胞亚群具有较贴壁细胞更强的克隆形成能力和低剂量体内成瘤能力.细胞总RNA经体外扩增获得的mRNA,可作为核酸抗原.结论应用无血清悬浮细胞球培养法可以获得高含量乳腺癌干细胞.通过体外扩增方法获得该细胞亚群的mRNA,可作为肿瘤核酸抗原,为下一步制备靶向乳腺癌干细胞的树突细胞瘤苗奠定了基础.