壳聚糖衍生物的制备及抗新城疫病毒活性研究

发布时间：2018-06-06 16:25

本文选题：壳聚糖衍生物 + 不同构型　；参考：《中国科学院研究生院(海洋研究所)》2015年硕士论文

【摘要】：新城疫是由新城疫病毒(NDV)引起的禽类的一种以呼吸道、消化道黏膜出血为典型症状的高度接触性、急性败血性传染病。目前防治鸡新城疫的手段主要以预防为主,多采用疫苗方法。近年来大量研究表明,海洋生物多糖具有抗病毒和提高机体免疫力的活性。壳聚糖作为天然存在的唯一带正电荷的多糖,具有调节免疫、抗肿瘤、抗菌、降脂、保湿等非常好的生物活性。且研究发现,壳聚糖及其衍生物可抑制几十种植物病毒、类病毒的侵染,但其是否具有抗新城疫病毒活性,国内外还未见报道。本文首次以不同分子量、不同结构类型、不同基团修饰的α-壳聚糖、β-壳聚糖和6-胺取代-6-脱氧壳聚糖衍生物为原料,以鸡胚尿囊液中血凝效价的降低作为指标,针对不同样品抗鸡新城疫病毒活性进行了筛选;同时,采用荧光定量PCR技术,测定免疫因子基因表达的变化,研究壳聚糖衍生物对于免疫系统的激活与调控作用,从而确定壳聚糖衍生物对于机体免疫系统的影响及药效作用机制。具体结果如下:测试样品均具有抗新城疫病毒活性,分子量、分子结构、修饰基团、样品浓度和接种时间的差异对活性影响显著。1)α-壳聚糖和β-壳聚糖的抗新城疫病毒活性因分子量和浓度的不同而不同,最佳分子量分别为4513 Da和5743 Da,具有剂量依赖关系;2)β-壳聚糖的抗新城疫病毒活性明显优于α-壳聚糖,β-壳聚糖在分子量为3 kDa-6 kDa时,效果非常显著,可有效降低病毒血凝效价至0;3)6-脱氧-N-邻苯二甲酰基壳聚糖和6-脱氧-6-溴代-N-邻苯二甲酰基壳聚糖能够显著抑制新城疫病毒,可有效降低血凝效价至0,且效果好于低分子量α-壳聚糖及β-壳聚糖。荧光定量PCR测定结果表明:低分子量α-壳聚糖、β-壳聚糖能有效增强IL-2、IFN-α、IFN-β、TNF-α等免疫因子的基因表达,病毒RNA基因表达无明显变化。而经胺取代修饰的壳聚糖衍生物具有显著的抗新城疫病毒活性,同时有效调节免疫因子的基因表达,减少病毒RNA基因表达。本研究为获得一种绿色低毒、治疗有效的抗新城疫病毒类药物提供了可能。同时,也为我们系统性研究治疗其它免疫损伤性疾病奠定了基础。
[Abstract]:Newcastle disease (NDV) is a highly contact and acute septic infectious disease caused by Newcastle disease virus (NDV) in poultry with respiratory tract and gastrointestinal bleeding as typical symptoms. At present, the prevention and treatment of Newcastle disease are mainly based on prevention and vaccine. In recent years, a large number of studies have shown that marine polysaccharides have antiviral and immune activities. Chitosan, as the only positively charged polysaccharide, has very good biological activities such as immune regulation, anti-tumor, antibacterial, lipid-lowering, moisturizing and so on. It was found that chitosan and its derivatives could inhibit the infection of dozens of plant viruses and viroids, but whether they had the activity of resisting Newcastle disease virus had not been reported at home and abroad. In this paper, 伪 -chitosan, 尾 -chitosan and 6-amine substituted -6-deoxychitosan derivatives modified with different molecular weight, different structural types and different groups were used as raw materials for the first time. The reduction of hemagglutination titer in chicken embryo allantoic fluid was used as an index. The activity of different samples against Newcastle disease virus (NDV) was screened, and the changes of gene expression of immune factors were determined by fluorescence quantitative PCR, and the activation and regulation of chitosan derivatives on immune system were studied. The effects of chitosan derivatives on the immune system and the mechanism of pharmacodynamics were determined. The results are as follows: the tested samples have anti-Newcastle disease virus activity, molecular weight, molecular structure, modified group, The difference of sample concentration and inoculation time had a significant effect on the activity of 伪 -chitosan and 尾 -chitosan. The activity of anti-Newcastle disease virus was different with the molecular weight and concentration of 伪 -chitosan and 尾 -chitosan. The optimum molecular weight was 4513 Da and 5743 Da, respectively. The anti-Newcastle disease virus activity of 尾 -chitosan in a dose-dependent manner was significantly better than that of 伪 -chitosan. When the molecular weight of 尾 -chitosan was 3 kDa-6 kDa, the effect of 尾 -chitosan was very significant. The hemagglutination titer of the virus was reduced to 0 ~ (3) ~ (3) -deoxy-N-phthalyl chitosan and 6-deoxy-6-bromo-N-phthalyl chitosan, which could significantly inhibit Newcastle disease virus. The hemagglutination titer was reduced to 0, and the effect was better than that of low molecular weight 伪 -chitosan and 尾 -chitosan. The results of fluorescence quantitative PCR showed that low molecular weight 伪 -chitosan and 尾 -chitosan could effectively enhance the expression of IFN- 伪, IFN- 尾, TNF- 伪 and other immune factors, but the expression of virus RNA gene had no significant change. The chitosan derivatives modified by amines have significant anti-Newcastle disease virus activity and can effectively regulate the gene expression of immune factors and reduce the RNA gene expression of the virus. In this study, it is possible to obtain a green low-toxicity and effective anti-Newcastle disease virus drugs. At the same time, it also lays the foundation for systematic research on other immune damage diseases.
【学位授予单位】：中国科学院研究生院(海洋研究所)
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R943;R96

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