乳腺癌干细胞新型多肽标志物的筛选

目的:从噬菌体随机十二肽库中筛选出能够与乳腺癌干细胞(breast cancer stem cell,BCSC)特异性结合的噬菌体,筛选后提取多肽以研究其与BCSC的亲和力和特异性。方法:通过"无血清-有血清"交替培养法培养人乳腺癌MCF7和MDA-MB-231细胞系,以求最大化富集BCSC,将hs578bst正常人乳腺细胞和普通培养的乳腺癌细胞用作减性筛选细胞,筛选噬菌体随机肽库;然后根据筛选结果选取阳性噬菌体进行单克隆扩增并测序,得到测序结果后依据合成多肽,标记以FITC;最后,建立乳腺癌动物模型,并在体外观察合成多肽与BCSC结合的特异性。结果:经过3轮筛选,噬菌体富集约200倍;随机选出10株单克隆噬菌体,与BCSC共同培养,其中与MCF7和MDA-MB-231乳腺癌干细胞阳性结合的噬菌体数目分别为6株和8株;分别从中选取1个阳性噬菌体进行测序,合成多肽后分别命名为A3和B8;多肽A3特异性结合MCF7乳腺癌干细胞,同时多肽B8特异结合MDA-MB-231乳腺癌干细胞。结论:噬菌体随机肽库可成功筛选出能够特异性结合BCSC的多肽,为BCSC的靶向治疗和进一步研究奠定了理论基础。

Screening of novel specific polypeptide markers of breast cancer stem cells

Objective:To screen the phage specifically binding to breast cancer stem cells with normal breast cells and breast cancer cells as reduced screening control and identify the specificity，extracte the positive phage clones DNAs，synthesize the polypeptide according to the DNA sequence and labele FITC.Finally identify the specificity of the synthesized polypeptide in vitro.Methods:We cultured breast cancer stem cells and identified by flow cytometry.Phage random peptide library was amplified and screened by culturing with breast cancer cells and breast cancer stem cells.We identified the positive phage in ELISA and extracted the positive phage DNA.We isolated DNA pellet and sent them to biotechnology companies for sequencing.Then according to sequencing results，polypeptide was synthesized and labeled by FITC.Finally，the specificity to breast cancer stem cells was identified in vitro.Results:After three rounds of screening，the phage was enriched to about 200-fold.Immunofluorescence showed that randomly selected two phage clones B8 and A3 had specific affinity to the breast cancer stem cells.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 may lay the foundation for the targeted therapy and further study of breast cancer stem cells.