新型丙型肝炎病毒亲和吸附剂的制备及其应用的相关研究

发布时间：2019-01-30 20:58

【摘要】：丙型肝炎病毒是一种能引起重大肝脏类疾病的RNA病毒,严重威胁着人类的健康安全。病毒本身的遗传多变性导致了有效疫苗的研发困难,另外传统的药物治疗如干扰素治疗方法以及直接作用抗病毒药物面临治疗并发症、特定分型治疗差、部分病例难治愈以及成本高昂等问题。在这样的背景下,本研究针对HCV制备了一种新型的特异性亲和吸附剂。第一部分介绍了HCV病毒的分子生物学、遗传特征、治疗策略等基本情况,其次也对体外血液净化治疗的原理、分类、吸附剂载体、配体等方面进行了概括。其次,我们使用不同的交联方法制备亲和吸附剂并比较了包括偶联率、操作难易、安全环保、配体结合稳定性等方面,最终择优选取羧基化琼脂糖凝胶微球作为固相载体,EDC、NHS为交联剂并经过工艺优化制备了偶联率达91%的亲和吸附剂。随后对制备而成的亲和吸附剂进行电镜表征以及红外表征,验证功能配体成功偶联于固相载体之上。另外,我们研究了亲和吸附剂的生物相容性以及血液安全性,通过非特异性吸附、全血接触实验以及细胞毒性实验验证了这一点。我们进一步对亲和吸附剂的病毒吸附性能进行了考察,首先通过批量吸附实验,使用荧光定量PCR验证了亲和吸附剂对体外培养系统扩增的HCV的清除功效,其对病毒的清除率大约为89%,随后对亲和吸附剂清除病毒的时间特性进行了考察,荧光定量PCR技术以及Southern杂交等实验表明在2h内亲和吸附剂对目标样本的HCV清除率可达到85%以上。其次我们使用病人样本作为实验对象对亲和吸附剂的病毒清除性能再次进行验证,最后我们探讨了不同的灭菌工艺对亲和吸附剂的性能回复率影响。综上所述,我们使用功能核酸作为配体使用合适的方法成功制备了血液安全性良好的HCV特异性亲和吸附剂,其物理化学结构得到表征验证,同时其对体外培养系统或者病人样本HCV都具备良好的清除功效,应用之前合适的灭菌方法也得到确认,有望成为新型的HCV治疗策略。
[Abstract]:Hepatitis C virus (HCV) is a kind of RNA virus which can cause major liver diseases. It is a serious threat to human health and safety. The genetic variability of the virus itself makes it difficult to develop effective vaccines. In addition, traditional drug treatments, such as interferon treatment and direct antiviral drugs, face treatment complications, and specific types of treatment are poor. Some cases are difficult to cure and costly. In this context, a novel specific affinity adsorbent was prepared for HCV. In the first part, the molecular biology, genetic characteristics and therapeutic strategies of HCV virus were introduced. Secondly, the principle, classification, adsorbent carrier and ligand of in vitro blood purification therapy were also summarized. Secondly, we used different crosslinking methods to prepare affinity adsorbents and compared the coupling ratio, easy operation, safety and environmental protection, ligand binding stability and so on. Finally, carboxylated agarose gel microspheres were selected as solid phase carrier, EDC,. The affinity adsorbent with coupling efficiency of 91% was prepared by using NHS as crosslinking agent and optimized process. The affinity adsorbents were characterized by electron microscopy and IR, and the functional ligands were successfully coupled to the solid phase carrier. In addition, we studied the biocompatibility and blood safety of affinity adsorbent, which was verified by non-specific adsorption, whole blood contact test and cytotoxicity test. We further investigated the viral adsorption properties of affinity adsorbents. Firstly, batch adsorption experiments were carried out to verify the scavenging effect of affinity adsorbents on HCV amplified by culture system in vitro by using fluorescent quantitative PCR. The virus clearance rate was about 89%, and then the time characteristics of the affinity adsorbent to remove the virus were investigated. The results of fluorescence quantitative PCR and Southern hybridization showed that the HCV clearance rate of the affinity adsorbent to the target sample could reach more than 85% within 2 hours. Secondly, we used patient samples as experimental objects to verify the virus clearance performance of affinity adsorbent. Finally, we discussed the effect of different sterilization processes on the performance recovery rate of affinity adsorbent. In conclusion, we used functional nucleic acids as ligands to successfully prepare HCV specific affinity adsorbents with good blood safety, and their physical and chemical structures were characterized and verified. At the same time, it has good scavenging effect on in vitro culture system or HCV of patient sample, and the proper sterilization method before application is confirmed, which is expected to be a new treatment strategy for HCV.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R943

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