基于抗精子适体的新型外用避孕药物的基础研究

Family planning is China's basic national policy. Among numerous contraceptive approaches, much attention has been paid to vaginal spermicides because these products can be easily applied topically and do not interfere with endocrine function. However, the acceptability of the existing spermicides in the market has been constrained by the side effects including irritation, vaginal flora imbalance and genital ulceration. Aptamers are oligonucleotides with antibody-like features and can be selected from a synthesized random-sequence pool by a method called SELEX. However, it is hard to predict the selection outcome. The uncertainty of the method is due to its intrinsic imperfection: randomicity and size limitation of the initial sequence pool. Based on our expertise in development of aptamer, we propose to break through this common technical bottleneck by development of an in vitro directed evolutionary process towards generating desired sperm-binding sequence, thereby a novel approach for preparation of aptamer will be established. Furthermore, functional selection for candidate sperm-immobilizing sequences will be performed. In order to ensuring that aptamers against human sperm will be generated with the characteristics of not only strong affinity and high specificity but also high inhibition efficiency, the contraceptive potential of each individual monoclonal aptamers will be examined by a series of tests, including inhibition of acrosome reaction and zona-free hamster oocyte penetration. Eventually the best sequence of the aptamer will be identified. It is expected that the outcome of this study will add to our knowledge of specific inhibition of sperm function by a non-antibody reagent and open a new avenue for the development of a novel contraceptive drug.

计划生育是我国基本国策，外用杀精剂因具有操作方便、不干扰内分泌功能等优点而受到重视。但现有杀精剂存在的局部刺激、长期应用引发阴道菌群失调、阴道上皮损伤等缺点，限制了其广泛应用。适体是具有抗体功能特点的短链核酸分子，可以通过SELEX技术从人工合成的随机序列文库中筛选而获得。但能否筛选出理想序列却无法事先预估。这是由于起始文库的随机性和局限性致使筛选结果的不确定性。本课题组在多年研究适体药物的基础上，拟以人精子为靶标，通过体外分子定向进化途径突破这一关键共性技术瓶颈，从而建立一个制备适体的新的技术平台。在这基础上针对候选序列抑制精子的活动进行功能性筛选，再通过包括顶体反应和精卵融合的功能测试，进行甄别。从而获得亲和力强特异性高而且抑精功能确实的抗人精子单克隆适体。本课题通过基础研究探讨非抗体药物特异性抑精功能以实现避孕的作用，将有助于填补适体抑精方面的知识空白，为开发新型避孕药物奠定基础。

目前常见的杀精剂多是由表面活性剂为主要成分。这种杀精剂（例如含壬苯醇醚-9）可能出现局部烧灼感和分泌物增多的现象，甚至发生生殖道溃疡。多年来许多试验室提出了不同的新型杀精剂，例如：植物提取物，细菌成分，抗体，以及合成化合物。其中除了抗体，其他方法都不具备精子特异性，对非精子细胞也同时具有不同程度的损伤作用，更有甚者，未被完全杀灭的精子留下致畸的隐患。应用抗体虽然可以特异性地与精子结合而使其失能，但由于成本高、不易储存和使用，至今无法推广。适体是能够与靶标特异性结合的单链DNA或RNA，也被称为“化学抗体”，迄今尚未见抗人精子适体应用于体外避孕方面的报道。本课题组以人精子细胞为靶标，制备抗人精子适体。.本项目已经按照原计划圆满完成研究任务。主要进展包括：①通过对原有技术SELEX（Systematic Evolution of Ligands by EXponential enrichment指数富集配体系统进化）的改良，克服了起始文库和筛选结果的不确定性，建立了SELEM（Systemic Evolution of Ligands by Enhanced Mutation-selection强诱变配体系统进化）体外分子定向进化新方法，突破了关键共性技术瓶颈，保证适体序列的强亲和性。②通过阴道上皮细胞对动态文库进行反筛，保证适体序列的高特异性。避免事先了解精子细胞表面乃至靶标分子结构细节的繁琐复杂工作。③通过功能性筛选，留取明显抑制精子活力但不影响精子存活率的适体序列。应用这种适体开发外用避孕药，既能避免受精机会又消除了致畸的根源。④把两个适体序列通过短链连接成为二价适体。利用计算机辅助精子分析系统（computer aided sperm analysis, CASA）分析处理，证明适体可以明显抑制精子活力，特别是促进精子凝集，达到了原定的目标，填补了适体抑精方面的知识空白，为开发新型避孕药物奠定基础。.本课题的部分内容与新西兰皇家科学院合作研究，发表的期刊论文以中国国家自然科学基金为第一标注经费支持者，以中国国立华侨大学为第一作者单位，以本课题负责人为第一作者兼通讯作者（一共6名作者）。.通过本课题的研究，发表了8篇期刊论文、申请了1项专利、培养7名研究生。