宫颈癌HeLa细胞特异性结合肽的筛选及鉴定

Screening and identification of the peptides specifically binding to cervical cancer HeLa cell

背景与目的:宫颈癌的分子靶向治疗具有很好的疗效,同时可以显著减少抗癌药物对人体自身的损伤,因此备受关注。本研究利用噬菌体体内展示技术筛选及鉴定宫颈癌特异性结合肽,将有可能成为化疗药物的靶向载体,为宫颈癌靶向药物治疗奠定基础。方法:体外培养宫颈癌HeLa细胞接种裸鼠,建立肿瘤动物模型。将随机肽库尾静脉注入裸鼠体内,循环15 min,心脏灌注后回收肿瘤组织噬菌体扩增、纯化并以此作为起始物进行第2轮的筛选,如此进行3轮体内筛选后挑取噬菌体克隆,进行免疫组化及ELISA实验,初步鉴定噬菌体克隆对宫颈癌细胞的亲和力及特异性,并将具有强亲和力的克隆进行测序。结果:ELISA结果显示,随机挑选10个噬菌体单克隆中8个克隆对HeLa细胞具有很强的亲和力,将这8个克隆进行测序,获得相同短肽序列LLRSTGF。结论:利用噬菌体展示技术筛选出与宫颈癌细胞HeLa特异性结合的短肽,进一步与化疗药物结合,为宫颈癌靶向治疗提供新的方法。

Background and purpose： Molecular targeted therapy was paid much attention for its good therapeutic effects on cervical cancer and deeply decreased the injuries of the human body. This study aimed to screen and identify the peptides specifically binding to cervical cancer by using phage display in vivo. It may be the targeting vector of the chemotherapeutics and be the basic of the targeted drugs of cervical cancer. Methods： Human cervical cancer HeLa cell was cultured in vivo and the tumor animal model was made in nude mice. After phage peptide library was injected in mouse by intravenous injection and heart perfusion was carried out after 15 minutes, cervical cancer was harvested. Cervical cancer specific peptides that used in the next screening were obtained after amplification and purification in vivo. After 3 circulations, the affinity and specificity of phage clones to cervical cancer cells were preliminary indentified by enzyme linked immunosorbent assay （ELISA） and immunohistochemistry. The positive phage clone was amplified and the sequences were analyzed to determine the collective sequence. Results： The 10 phage clones were identified by ELISA, 8 of them were specially bound to the HeLa cell line which had the same amino acid sequence that is LLRSTGF. Conclusion： The peptides specifically binding to cervical cancer HeLa cell are screened and identified. All the results lay the foundation for the development of diagnostic reagents and drugs of cervical cancer.