人乳头瘤病毒 (HPV) 16型E7蛋白特异性亲和体分子靶向治疗宫颈癌的作用及机制研究

Molecular targeted therapy for various cancers based on antibodies has shown its unique advantage and application prospect. Affibody molecules, a class of small single domain proteins that can be isolated for high affinity and specificity to any given protein target, are recently proven to have greater potential in targeted therapy for cancers than antibodies. We have obtained HPV 16 E7-specific affibody molecule by phage display technology. This affibody molecule can kill cervical cancer cells by binding to HPV16 E7 with high affinity and specificity while the detailed molecular mechanism is not clear. In this study, based on our previous results, we try to explore the effect of the HPV 16 E7-specific affibody molecule on signal transduction pathways such as pRB/p53, PI3K/Akt/mTOR, and JAK/STAT. Specifically, we will analyze the expression level of several vital proteins including pRB, E2F1, Ras, Notch, PIK3CA and STAT5 with methods and assays such as real-time PCR、western blot, flow cytometry, and so on. Then, we will generate the xenograft tumor mouse model for human cervical cancer and confirm the mechanism of its cervical cancer cell killing function both in vitro and in vivo. The data accumulated in this study will serve as a foundation for the targeted therapy for human cervical cancer with affibody molecules.

基于抗体的肿瘤分子靶向治疗已显示了其独特的优势和应用前景，而来源于葡萄球菌蛋白A的亲和体(affibody)分子，能与靶蛋白高亲和力结合，且组织穿透性良好，应用于肿瘤靶向治疗比抗体更具优势。本研究团队利用噬菌体展示技术，筛选到了能够特异靶向结合HPV 16肿瘤原性蛋白E7，并杀伤宫颈癌细胞的affibody分子。本项目拟在前期研究基础上，通过real-time PCR、western blot和流式细胞术等方法，分析E7作用相关的pRB、E2F1、Ras、Notch、PIK3CA和STAT5等因子的表达变化，探讨该affibody分子对pRB/p53、PI3K/Akt/mTOR和JAK/STAT等信号通路的影响，在细胞水平验证其对宫颈癌细胞杀伤作用的分子机制，并通过宫颈癌细胞荷瘤裸鼠模型进一步从动物水平进行验证。为基于affibody分子的宫颈癌特异性靶向治疗提供理论基础和实验依据。

人乳头瘤病毒(human papillomavirus, HPV) 16、18等高危型别感染与宫颈癌的发生与发展的关系已经明确。虽然HPV疫苗已成功上市用于预防宫颈癌，但是，对于已经感染HPV及其所致的癌前病变和宫颈癌的治疗，至今仍没有特异方法。宫颈癌死亡率在全球妇女癌症中高居第二甚至首位[1]，我国宫颈癌每年新发病例为13万例以上，约有5万人死于宫颈癌。由HPV感染所致的宫颈癌已对我国妇女健康构成了极大危害。因此，HPV感染所致宫颈癌的特异性治疗研究已经迫在眉睫。.基于抗体的肿瘤分子靶向治疗已显示了其独特的优势和应用前景，而来源于葡萄球菌蛋白A的亲和体(affibody)分子，能与靶蛋白高亲和力结合，且组织穿透性良好，应用于肿瘤靶向治疗比抗体更具优势。本项目在本研究团队利用噬菌体展示技术，筛选到了能够特异靶向结合HPV 16肿瘤原性蛋白E7，并杀伤宫颈癌细胞的affibody分子的前期基础上，对筛选的HPV16 E7 affibody分子进入靶细胞以及对靶细胞杀伤作用的机制进行了研究，即HPV16 E7 affibody分子对表达HPV16 E7蛋白的宫颈癌细胞（以下简称为HPV16 E7阳性宫颈癌细胞）的杀伤作用验证、进入靶细胞的机制、发挥作用的信号通路等进行了系统研究。