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：

噬菌體展示; 喉癌細胞; 靶向性

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【摘要】　目的　篩選人源喉癌Hep-2細胞株特異結合的短肽，作為喉癌靶向治療的載體。　方法　體外培養Hep-2細胞株作為靶細胞，人正常喉黏膜上皮細胞為吸附細胞；用噬菌體展示十二肽庫進行3輪差減篩選，隨機挑取10個噬菌體克隆進行測序；采用酶聯免疫吸附（enzyme linked immunosorbent assay，ELISA）法鑒定噬菌體與Hep-2細胞的結合活性；通過免疫熒光鑒定喉癌細胞特異性結合肽（F2）噬菌體陽性克隆與喉癌細胞結合的特異性。　結果　經過3輪篩選后，噬菌體在靶細胞Hep-2上出現明顯富集；ELISA分析鑒定顯示5個陽性克隆能與Hep-2細胞特異結合，其中F2噬菌體克隆對喉癌細胞的結合靶向性明顯高于對照細胞（P lt;0.05）；免疫熒光顯色顯示，F2能特異性地與喉癌細胞結合。　結論　利用噬菌體展示肽庫技術，可以成功篩選到F2，其可能成為喉癌靶向治療的載體。
【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. 　柯朝陽, 吳展元. 人喉上皮細胞的體外培養及生物學特性[J]. 廣東醫學, 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, 姚學清, 吳承堂, 等. 噬菌體展示肽庫篩選大腸癌細胞特異性結合肽的實驗研究[J]. 南方醫科大學學報, 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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