Ph.D.-7TM肽库体内淘选卵巢癌A2780细胞特异性结合肽
In Vivo Screening with Ph.D.-7TM and Identification of Ovarian Cancer A2780 Cell Targeted Heptapeptides

作者: 李钰璨 , 尹光福 , 蒲曦鸣 , 尤 飞 :四川大学材料科学与工程学院,四川 成都;

关键词: 卵巢癌A2780细胞体内淘选噬菌体展示靶向七肽Ovarian Cancer A2780 Cells In Vivo Biopanning Phage-Displayed Peptide Library Targeting Heptapeptide

摘要:
卵巢癌是女性生殖器官常见的恶性肿瘤之一,卵巢上皮癌死亡率居各类妇科肿瘤的首位。探索高效安全的治疗手段,已经成为卵巢癌治疗领域的热点问题。目的:应用噬菌体展示技术体内筛选、鉴定并评价卵巢癌特异性结合七肽,为卵巢癌靶向给药系统的构建及肿瘤靶向诊疗技术的研究奠定基础。方法:噬菌体展示七肽库Ph.D.-7TM在卵巢癌A2780荷瘤模型体内进行3轮淘选。经ELISA、体内外结合、结合抑制和免疫荧光试验鉴定肿瘤阳性噬菌体单克隆及其对应展示肽的特异性,MTT试验、划痕试验考察靶向肽对相关细胞行为的影响。结果:测序结果中表达HGGVRLY肽(HGG肽)的噬菌体单克隆PC-H所占比例达到27%,ELISA中PC-H对A2780细胞显示出高亲和力及显著特异性(P < 0.05)。体内、体外反筛结果表明PC-H对A2780细胞和肿瘤组织具有很强亲和力。结合抑制实验及荧光显微成像证明了HGG肽对A2780细胞的靶向性,且HGG肽对肿瘤细胞的行为无显著影响,对正常细胞无抑制。结论:应用体内噬菌体肽库技术成功筛选出可与卵巢癌A2780细胞特异性结合的靶向肽HGGVRLY,其细胞相容性良好,可用于卵巢癌的诊断和靶向治疗。

Abstract: In order to select the specific ligands for ovarian cancer, the in vivo phage display technology has been applied to screen the peptide targeting to ovarian cancer A2780 cells. Firstly, ovarian cancer A2780 cells were inoculated into the armpit of nude mice to establish a tumor-bearing animal model. Then in vivo biopanning was conducted for 3 rounds with the Ph.D.-7TM phage-displayed peptide library, and individual phage clones were picked for DNA sequencing after the 3rd round. Among the picked phage clones, the clone PC-H displaying heptapeptide HGGVRLY (peptide HGG) accounted for as high as 27 percent. The identification results by ELISA tests, in vivo and in vitro phage binding assays, competitive binding assays, and immunofluorescence assays indicated that both the phage clone PC-H and the peptide HGG could be preferentially bounded to A2780 cells and then concentrated in tumor tissues. Furthermore, the results of MTT assay and scratch assay revealed that peptide HGG possessed low cytotoxicity. Altogether, the peptide HGG screened with the Ph.D.-7TM exhibited the high affinity to A2780 cells and the targeting to the ovarian cancer tis-sues, and would have great potentials in the targeting drug delivery system for ovarian cancer.

文章引用: 李钰璨 , 尹光福 , 蒲曦鸣 , 尤 飞 (2015) Ph.D.-7TM肽库体内淘选卵巢癌A2780细胞特异性结合肽。 世界肿瘤研究, 5, 43-51. doi: 10.12677/WJCR.2015.53007

参考文献

[1] International Agency for Research on Cancer (2014) World cancer report 2014. In: Stewart, B.W. and Wild, C.P., Eds., WHO Press, Lyon, 16-53.

[2] Auersperg, N., Wong, A.S.T., Choi, K.C., et al. (2001) Ovarian surface epithelium: Biology, endocrinology, and pathology. Endocrine Reviews, 22, 255-288.

[3] Marchetti, C., Pisano, C., Facchini, G., Bruni, G.S., Magazzino, F.P., Losito, S. and Pignata, S. (2010) Fist-line treatment of ovarian cancer: Current research and perspectives. Expert Review, 10, 47-60.
http://dx.doi.org/10.1586/era.09.167

[4] Hajitou, A., Pasqualini, R. and Arap, W. (2006) Vascular targeting: Recent advances and therapeutic perspectives. Trends in Cardiovascular Medicine, 16, 80-88.
http://dx.doi.org/10.1016/j.tcm.2006.01.003

[5] Kigawa, J. (2013) New Strategy for Overcoming Resistance to Chemotherapy of Ovarian Cancer. Yonago Acta Medica, 56, 43-50.

[6] Janczar, S., Graham, J.S., Paige, A.J.W. and Gabra, H. (2009) Targeting locoreginal peritoneal dissemination in ovarian cancer. Expert Review of Obstetrics & Gy-necology, 4, 133-147.
http://dx.doi.org/10.1586/17474108.4.2.133

[7] Lafky, J.M., Wilken, J.A., Baron, A.I., et al. (2008) Clinical implications of the ErbB/epidermal growth factor (EGF) receptor family and its ligands in ovarian cancer. Biochimica et Biophysica Acta-Reviews on Cancer, 1785, 232-265.
http://dx.doi.org/10.1016/j.bbcan.2008.01.001

[8] Wilken, J.A., Badri, T., Cross, S., et al. (2012) EGFR/HER-targeted therapeutics in ovarian cancer. Future Medicinal Chemistry, 4, 447-469.
http://dx.doi.org/10.4155/fmc.12.11

[9] Xia, B., Yang, S., Liu, T., et al. (2015) miR-211 suppresses epithelial ovarian cancer proliferation and cell-cycle progression by targeting Cyclin D1 and CDK6. Molecular Cancer, 14, 57.
http://dx.doi.org/10.1186/s12943-015-0322-4

[10] Zhang, H.Y., Zhang, X.L., Ji, S.F., et al. (2014) Sohlh2 inhibits ovarian cancer cell proliferation by up regulation of p21 and down regulation of cyclin D1. Carcinogenesis, 35, 1863-1871.
http://dx.doi.org/10.1093/carcin/bgu113

[11] Yang, Z.Y., Zhou, X.S., Liu, Y., et al. (2014) Activation of integrin beta 1 mediates the increased malignant potential of ovarian cancer cells exerted by inflammatory cytokines. Anti-Cancer Agents in Medicinal Chemistry, 14, 955-962.
http://dx.doi.org/10.2174/1871520614666140613123108

[12] Adams, G.P. and Weiner, L.M. (2005) Monoclonal antibody therapy of cancer. Nature Biotechnology, 23, 1147-1157.
http://dx.doi.org/10.1038/nbt1137

[13] Brown, K.C. (2010) Peptidic tumor targeting agents: the road from phage display peptide selections to clinical applications. Current Pharmaceutical Design, 16, 1040-1054.
http://dx.doi.org/10.2174/138161210790963788

[14] Akita, N., Maruta, F., Seymour, L.W., Kerr, D.J., Parker, A.L., et al. (2006) Identification of oligopeptides binding to peritoneal tumors of gastric cancer. Cancer Science, 97, 1075-1081.
http://dx.doi.org/10.1111/j.1349-7006.2006.00291.x

[15] Chen, B., Cao, S., Zhang, Y., et al. (2009) A novel pep-tide (GX1) homing to gastric cancer vasculature inhibits angiogenesis and cooperates with TNF alpha in anti-tumor therapy. BMC Cell Biology, 10, 63.
http://dx.doi.org/10.1186/1471-2121-10-63

[16] Du, B., Han, H., Wang, Z., et al. (2010) Targeted drug delivery to hepatocarcinoma in vivo by phage-displayed specific binding peptide. Molecular Cancer Research, 8, 135-144.
http://dx.doi.org/10.1158/1541-7786.MCR-09-0339

[17] Laakkonen, P., Porkka, K., Hoffman, J.A., et al. (2002) A tumor-homing peptide with a targeting specificity related to lymphatic vessels. Nature Medicine, 8, 751-755.
http://dx.doi.org/10.1038/nm720

[18] Arap, W., Pasqualini, R. and Ruoslahti, E. (1998) Cancer treatment by tar-geted drug delivery to tumor vasculature in a mouse model. Science, 279, 377-380.
http://dx.doi.org/10.1126/science.279.5349.377

[19] Kalir, T., Firpo-Betancourt, A. and Nezhat, F. (2013) Update on ovarian cancer patho-genesis: History, controversies, emerging issues and future impact. Expert Review of Obstetrics & Gynecology, 8, 539-547.
http://dx.doi.org/10.1586/17474108.2013.847638

[20] Hajitou, A., Trepel, M., Lilley, C.E., et al. (2006) A hybrid vector for ligand-directed tumor targeting and molecular imaging. Cell, 125, 385-398.
http://dx.doi.org/10.1016/j.cell.2006.02.042

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