基于核酸适体技术的清开灵注射液不良反应的物质基础研究

The adverse reactions caused by the traditional Chinese medicine has become the focus of doctors and researchers，especially the Traditional Chinese Medicine Injections. They are multi-component systems. The chemical components which trigger the side effects are under discussion. The target component knockout is a systematical strategy to study the function of one certain fraction in the complicated Chinese Medicine. An aptamer is like an antibody, and it has the advantages of easily preparation, various ligands, stability, no Immunogenicity and et al. Therefore it can be applied to remove the specific constituent from tradition Chinese medicine in order to clarify its role in the complex. . The allergic reactions caused by Qingkailing Injection usually happened when applied to the patients. In this project, the Qingkailing Injection is used as the material. We employ the strategy of target-knockout to obtain the modified Qingkailing Injection. The aptamer technology and biological magnetic bead separation help to get rid of the specific constituent of the injection complex. The baicalin, geniposide and chlorogenic acid are the possible chemicals which cause the allergic reactions according the literatures. Therefore the baicalin-, geniposide- and chlorogenic acid-knockout Qingkailing Injections are prepared. The anaphylactic and allergic assays are conducted with the modified injections. The active fraction which caused the adverse reactions will be uncovered by comparison the results of Qingkailing Injection group and the target knockout group. In addition, the effects of the injection group and the knockout group on the brain ischemia rat model are also investigated. The relationship between toxicology and efficacy of each test component is evaluated and the component which caused the adverse reactions is expected to be identified in the Qingkailing Injection. Through the project, the technology platform with the aptamer and biomagnetic bead isolation method will be established to study the functions of chemicals in the Traditional Chinese Medicine injections which may produce the adverse reactions..

中药为复杂的多组分体系，其引发的不良反应，成为医学界共同关注的焦点。目前关于中药不良反应的物质基础尚不明确。基于目标成分剔除策略，将特定成分从中药组分中除去，能够系统的研究该成分在中药复杂体系中的作用。核酸适体是一种功能类似抗体的寡聚单链核苷酸，具有制备方便、靶分子范围广、稳定性高、无免疫原性等优势，能够实现上述目标。清开灵注射液在临床上引起的过敏反应最为常见，本项目以清开灵注射液为研究对象，建立基于核酸适体的不良反应物质基础研究平台，通过指数富集配基系统进化技术体外筛选与清开灵注射液可疑致敏原（黄芩苷、栀子苷和绿原酸）特异结合的核酸适体，采用适体固定化和磁珠分离技术，获得目标成分剔除后的清开灵注射液。通过研究其过敏反应的性质，结合脑缺血药效模型的作用，综合评价上述三种成分的毒效关系，明确清开灵注射液中不良反应的物质基础，为其临床合理用药、预防不良反应提供科学依据。

中药为复杂的多组分体系，其引发的不良反应以及中药不良反应的物质基础等，已成为医学界共同关注的焦点。本研究以清开灵注射液为研究对象，利用SELEX技术，制备可疑致敏原的核酸适配体，并通过核酸适体与可疑致敏原特异性结合，获得致敏成分剔除后的清开灵注射液，研究其不良反应的物质基础。研究主要包括三部分：一，将标记有生物素的核酸适配体与包被有链霉亲和素的磁珠偶联，将偶联物与清开灵注射液反应孵育，然后利用磁分离技术剔除其中的目标分子，如胆酸、栀子苷，获得剔除后的清开灵注射液。二，利用RBL-2H3细胞进行类过敏反应实验，对比剔除部分目标分子前后的清开灵注射液对细胞的类过敏作用，从而确定目标分子是否在清开灵注射液致类过敏反应中起作用。三，利用大鼠全身主动过敏实验和皮肤被动过敏反应模型，对比剔除部分目标分子前后的清开灵注射液的致敏作用，进一步验证目标分子是否在清开灵注射液的致过敏反应中起作用。