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目的筛选人源喉癌Hep-2细胞株特异结合的短肽,作为喉癌靶向治疗的载体。方法体外培养Hep-2细胞株作为靶细胞,人正常喉黏膜上皮细胞为吸附细胞;用噬菌体展示十二肽库进行3轮差减筛选,随机挑取10个噬菌体克隆进行测序;采用酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)法鉴定噬菌体与Hep-2细胞的结合活性;通过免疫荧光鉴定喉癌细胞特异性结合肽(F2)噬菌体阳性克隆与喉癌细胞结合的特异性。结果经过3轮筛选后,噬菌体在靶细胞Hep-2上出现明显富集;ELISA分析鉴定显示5个阳性克隆能与Hep-2细胞特异结合,其中F2噬菌体克隆对喉癌细胞的结合靶向性明显高于对照细胞(P<0.05);免疫荧光显色显示,F2能特异性地与喉癌细胞结合。结论利用噬菌体展示肽库技术,可以成功筛选到F2,其可能成为喉癌靶向治疗的载体。
Objective To screen short peptides specific for human laryngeal carcinoma Hep-2 cell line as a targeted therapy for laryngeal cancer. Methods Hep-2 cells were cultured in vitro as target cells and normal human laryngeal mucosa epithelial cells as adherent cells. The phage displayed twelve-peptide library for three rounds of subtractive screening, and 10 phage clones were randomly selected for sequencing. Enzyme-linked immunosorbent assay The binding activity of phage to Hep-2 cells was identified by enzyme-linked immunosorbent assay (ELISA). The specificity of phage-positive clones expressing laryngocarcinoma cell-specific binding peptide (F2) and laryngocarcinoma cells was identified by immunofluorescence. Results After 3 rounds of screening, the phages displayed obvious enrichment on target Hep-2 cells. ELISA analysis showed that 5 phage clones could specifically bind to Hep-2 cells. (P <0.05). Immunofluorescence staining showed that F2 could specifically bind to laryngeal carcinoma cells. Conclusion The phage displayed peptide library technology can successfully screen F2, which may become the carrier of targeted therapy for laryngeal cancer.