Liu Tingting, Wang Hongyue, Liu Zhiyong, Zhang Jing, Liu Yan, Zhang Lin, Zheng Chunhui, Liu Fei, Hou Chuanqiang, Li Baojiang
Department of Breast Surgery, Breast Cancer Center, Tai'an Central Hospital, Tai'an, China.
Department of Central Sterile Supply, Tai'an Central Hospital, Tai'an, China.
Front Oncol. 2021 May 28;11:647291. doi: 10.3389/fonc.2021.647291. eCollection 2021.
We screened the TNBC stem cells using phage display (PD) and acquired the specific binding clones; and then the positive phage DNAs were amplified and extracted, synthesized with specific polypeptides, and labeled with fluorescein isothiocyanate (FITC). Finally, we identified the specificity of the polypeptides and . Human breast cancer cell line MDA-MB-231 and human mammary gland cell line hs578bst were chosen in our study, and MDA-MB-231 breast cancer stem cells (BCSCs) were cultured and identified by flow cytometry. The phage peptide library was screened using MDA-MB-231 BCSCs, the positive phage clones were identified by ELISA, and the DNA of the positive phages was extracted and sent to a biotechnology company for sequencing. According to the sequencing results, a specific polypeptide was synthesized and labeled with FITC. In the end, the specificity of a polypeptide to BCSCs was identified and . The MDA-MB-231 BCSCs were cultured and enriched with the "serum and serum-free alternate" method. The BCSCs were found to have characteristics of CD44/CD24 epithelial surface antigen (ESA) and ALDH with flow cytometry. The phage was enriched to 200-fold after three rounds of screening for MDA-MB-231 BCSCs. The positive phages were sequenced; then a polypeptide named M58 was synthesized according to sequencing results. Polypeptide M58 has a specific affinity to MDA-MB-231 BCSCs and . Specific polypeptides binding to MDA-MB-231 BCSCs were screened out by PD screening method, which laid a theoretical foundation for the targeted therapy and further research of BCSCs.
我们使用噬菌体展示（PD）筛选三阴性乳腺癌干细胞并获得特异性结合克隆；然后扩增并提取阳性噬菌体DNA，与特异性多肽合成，并用异硫氰酸荧光素（FITC）标记。最后，我们鉴定了多肽的特异性。本研究选用人乳腺癌细胞系MDA-MB-231和人乳腺细胞系hs578bst，并通过流式细胞术培养和鉴定MDA-MB-231乳腺癌干细胞（BCSCs）。使用MDA-MB-231 BCSCs筛选噬菌体肽库，通过酶联免疫吸附测定（ELISA）鉴定阳性噬菌体克隆，并提取阳性噬菌体的DNA，送至生物技术公司进行测序。根据测序结果，合成一种特异性多肽并用FITC标记。最后，鉴定了一种多肽对BCSCs的特异性。采用“血清与无血清交替”法培养和富集MDA-MB-231 BCSCs。通过流式细胞术发现BCSCs具有CD44/CD24上皮表面抗原（ESA）和乙醛脱氢酶（ALDH）的特征。对MDA-MB-231 BCSCs进行三轮筛选后，噬菌体富集了200倍。对阳性噬菌体进行测序；然后根据测序结果合成一种名为M58的多肽。多肽M58对MDA-MB-231 BCSCs具有特异性亲和力。通过PD筛选方法筛选出与MDA-MB-231 BCSCs结合的特异性多肽，为BCSCs的靶向治疗和进一步研究奠定了理论基础。
