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目的:无血清培养法富集乳腺癌干细胞(breast cancer stem cell,BCSC)并采用噬菌体展示技术,筛选能特异性结合乳腺癌干细胞的噬菌体多肽。方法:无血清培养法富集乳腺癌MDA-MB-231细胞株中干细胞并以此为靶标,以hs578bst人正常乳腺细胞及普通培养的MDA-MB-231细胞为减性筛选细胞,对噬菌体随机肽库进行双重减性筛选,选取富集后的阳性噬菌体单克隆,ELISA及DAB染色鉴定阳性噬菌体特异性并测序。结果:经过3轮筛选,噬菌体得到约500倍的富集,随机挑选10株单克隆噬菌体。ELISA显示,6号噬菌体单克隆对乳腺癌干细胞的亲和力是对照的6.14倍;DAB鉴定亦显示,其对乳腺癌干细胞的特异性及亲和力最高,对阳性噬菌体DNA测序翻译得到十二肽氨基酸为GYSASRSTIPGK。结论:通过干细胞富集及噬菌体展示技术,成功筛选出能够特异性结合乳腺癌干细胞的特异性噬菌体多肽,为乳腺癌的干细胞靶向治疗和深入研究奠定基础。
OBJECTIVE: To enrich breast cancer stem cells (BCSC) by serum-free culture and to screen for phage polypeptides that specifically bind breast cancer stem cells using phage display technology. Methods: The serum-free culture method was used to enrich the stem cells of breast cancer cell line MDA-MB-231. The hs578bst human normal breast cells and the normal MDA-MB-231 cells were screened for selection. Peptide library was screened by double subtraction, and the positive phage clones after enrichment were selected. The positive phages were identified by ELISA and DAB staining and sequenced. Results: After 3 rounds of screening, the phage was enriched about 500-fold and randomly selected 10 monoclonal phages. ELISA showed that the phage No. 6 monoclonal antibody affinity to breast cancer stem cells is 6.14 times the control; DAB also showed that the specificity and affinity of breast cancer stem cells highest, positive phage DNA sequencing translated dodecapeptide amino acid GYSASRSTIPGK . Conclusion: The specific phage polypeptides that can specifically bind to breast cancer stem cells were successfully screened through stem cell enrichment and phage display technology, which laid the foundation for the targeted therapy and further study of breast cancer stem cells.