馮俊 1,2 , 李麗 3 , 楊洪斌 2 , 劉世喜 1
  • 1 四川大學華西醫(yī)院耳鼻咽喉頭頸外科（成都，610041）;2 川北醫(yī)學院附屬醫(yī)院耳鼻咽喉頭頸外科;3 川北醫(yī)學院病理教研室;

【摘要】　目的　 篩選人源喉癌Hep-2細胞株特異結(jié)合的短肽，作為喉癌靶向治療的載體。　方法　 體外培養(yǎng)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(lián)免疫吸附（enzyme linked immunosorbent assay，ELISA）法鑒定噬菌體與Hep-2細胞的結(jié)合活性；通過免疫熒光鑒定喉癌細胞特異性結(jié)合肽（F2）噬菌體陽性克隆與喉癌細胞結(jié)合的特異性。　結(jié)果　 經(jīng)過3輪篩選后，噬菌體在靶細胞Hep-2上出現(xiàn)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(jié)合，其中F2噬菌體克隆對喉癌細胞的結(jié)合靶向性明顯高于對照細胞（P lt;0.05）； 免疫熒光顯色顯示，F(xiàn)2能特異性地與喉癌細胞結(jié)合。　結(jié)論　 利用噬菌體展示肽庫技術(shù)，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.

引用本文： 馮俊,李麗,楊洪斌,劉世喜. 用噬菌體展示技術(shù)篩選喉癌細胞靶向肽的研究. 華西醫(yī)學, 2011, 26(12): 1844-1847. doi: 復制

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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.
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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.
  1. 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. 2. 　Smith RB. Surgery in the management of laryngeal and hypopharyngeal carcinoma [J]. Int J Radiat Oncol, 2007, 69(Suppl 2): 28-30.
  3. 3. 　柯朝陽, 吳展元. 人喉上皮細胞的體外培養(yǎng)及生物學特性[J]. 廣東醫(yī)學, 2005, 26(6): 798-800.
  4. 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. 5. 　Parmley SF, Smith GP. Antibody-selectable filamentous phage vectors. affinity purification of target genes[J]. Gene, 1988, 73(2): 305-318.
  6. 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. 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. 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. 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. 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(jié)合肽的實驗研究[J]. 南方醫(yī)科大學學報, 2008, 28(6): 986-989.
  11. 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. 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. 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. 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. 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.