用噬菌體展示技術篩選喉癌細胞靶向肽的研究_《華西醫學》

作者：

馮俊 ^1,2 , 李麗 ³ , 楊洪斌 ² ,  劉世喜 ¹

1 四川大學華西醫院耳鼻咽喉頭頸外科（成都，610041）;2 川北醫學院附屬醫院耳鼻咽喉頭頸外科;3 川北醫學院病理教研室;

關鍵詞：

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.

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

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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. 　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.

《華西醫學》

用噬菌體展示技術篩選喉癌細胞靶向肽的研究

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《華西醫學》

用噬菌體展示技術篩選喉癌細胞靶向肽的研究

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