Research on Screening Peptides Specifically Targeting Laryngeal Squamous Cell Carcinoma by Phage Display Technique_West China Medical Journal

Authors：

FENG Jun ^1,2 , LI li ³ , YANG Hongbing ² ,  LIU Shixi ¹

1. Department of Otolaryngology-Head and Neck Surgery, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China; 2. Department of Otolaryngology-Head and Neck Surgery, Affiliated Hospital of North Sichuan Medical College, Nanchong, Sichuan 637000, P. R. China; 3. Department of Pathology, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, P. R. China;

Corresponding author：

LIU Shixi, Email: flj20041201@163.com

Keywords：

噬菌体展示; 喉癌细胞; 靶向性

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【摘要】　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 lt;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。
【Abstract】　Objective　 To obtain the polypeptides specifically bound to laryngeal squamous cell carcinoma line (Hep-2) and use it as a potential therapeutic vector targeting laryngeal squamous cell carcinoma patients.　Methods　 With the Hep-2 cells as the target cells and human normal laryngeal squamous epithelial cells (HNLE cells) as the absorber cells, 3 rounds of panning from a Ph.D.-12TM phage-display peptide library were carried out. Ten randomly selected phage clones were sent for sequence detection. The affinity of phage clones was detected by enzyme-linked immunosorbent assay (ELISA). The positive phage clones (F2) specifically bound to Hep-2 were identified by immunofluorescence detection.　Results　 After 3 rounds of screening, 5 positive phage clones showed specific binding to Hep-2 cells and the affinity of positive phage clones (F2) was significantly higher than that of the control groups (P lt;0.05). The results of immunofluorescence detection indicated that F2 could be specifically bound to Hep-2.　Conclusions　 Phage display peptide libraries technique can successfully screen the peptide specifically bound to Hep-2 cell line. Thus, it provides a potential vector for targeting therapy of laryngeal squamous cell carcinoma patients.

Citation： FENG Jun,LI li,YANG Hongbing,LIU Shixi. Research on Screening Peptides Specifically Targeting Laryngeal Squamous Cell Carcinoma by Phage Display Technique. West China Medical Journal, 2011, 26(12): 1844-1847. doi: Copy

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1. 　Akman FC, Dag N, Ataman OU, et al. The impact of treatment center on the outcome of patients with laryngeal cancer treated with surgery and radiotherapy [J]. Eur Arch Otorhinolaryngol 2008, 265(10): 1245-1255.
2. 　Smith RB. Surgery in the management of laryngeal and hypopharyngeal carcinoma [J]. Int J Radiat Oncol, 2007, 69(Suppl 2): 28-30.
3. 　柯朝阳, 吴展元. 人喉上皮细胞的体外培养及生物学特性[J]. 广东医学, 2005, 26(6): 798-800.
4. 　Smith GP. Filamentous fusion phage. novel expression vectors that display cloned antigens on the virion surface[J]. Science, 1985, 228(4705): 1315-1317.
5. 　Parmley SF, Smith GP. Antibody-selectable filamentous phage vectors. affinity purification of target genes[J]. Gene, 1988, 73(2): 305-318.
6. 　David NK, Girja SS, Shen GP, et al. Selection of Tumor-binding Ligands in Cancer Patients with Phage Display Libraries[J]. Cancer Res, 2006, 66(15): 7724-7733.
7. 　Terada T, Inui K. Peptide transporters. structure, function, regulation and application for drug delivery[J]. Curr Drug Metab, 2004, 5(1): 85- 94.
8. 　Schluesener HJ, Tan X. Selection of recombinant phages binding to pathological endothelial and tumor cells of rat glioblastoma by in vivo display[J]. J Neurol Sci, 2004, 224(1-2): 77-82.
9. 　Reiersen H, Berntsen G, StassarM, et al. Screening human antibody libraries against carcinoma cells by affinity purification and polymerase chain reaction[J]. J Immunol Methods, 2008, 330(1-2): 44-56.
10. 　廖康雄, 姚学清, 吴承堂, 等. 噬菌体展示肽库筛选大肠癌细胞特异性结合肽的实验研究[J]. 南方医科大学学报, 2008, 28(6): 986-989.
11. 　Bing Yu, Ming Ni, Wen-Han Li, et al. Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library[J]. World J Gastroenterol, 2005, 11(26): 3985-3989.
12. 　Lee TY, Wu HC, Tseng YL, et al. A novel peptide specifically binding to nasopharyngeal carcinoma for targeted drug delivery[J]. Cancer Res, 2004, 64(21): 8002-8008.
13. 　Geuijen CA, BijlN, Smit RC, et al. A proteomic approach to tumor target identification using phage display, affinity purification and mass spectrometry[J]. Eur J Cancer, 2005, 41(1): 178-187.
14. 　Williams B, Atkins A, Zhang H, et al. Cell-based selection of internalizing fully human antagonistic antibodies directed against FLT3 for suppression of leukemia cell growth[J]. Leukemia, 2005, 19(8): 1432-1438.
15. 　Bakker AB, van den Oudenrijn S, BakkerA Q, et al. C-type lectin-like molecule-1: a novel myeloid cell surface marker associated with acute myeloid leukemia[J]. Cancer Res, 2004, 64(22): 8443-8450.

West China Medical Journal

Research on Screening Peptides Specifically Targeting Laryngeal Squamous Cell Carcinoma by Phage Display Technique

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