实验海洋生物学重点实验室知识库

甲壳素是储存量仅次于纤维素的天然海洋生物多糖，来源广泛。而壳聚糖是 甲壳素脱乙酰的产物，具有许多优良的生物特性，如天然无毒、生物相容性好、 抗病毒、免疫调节作用等。但其在水中的不溶性限制了其发展。而将壳聚糖进行 改性，在其链上引入新活性基团是提高壳聚糖水溶性，改善生物活性的有效手段 之一。在诸多衍生物中，季铵化可以增加壳聚糖的正电性、提高水溶性和生物活 性而备受关注。 课题组前期对壳聚糖季铵盐的免疫活性进行了研究，发现低分子量较高分子 量的壳聚糖季铵盐免疫活性高，而且可以激活 RAW264.7 细胞中的 MAPK， JAK/STAT 和 PI3K/Akt 通路，调节免疫应答。并发现将壳聚糖季铵盐制备成纳 米粒后能显著提高鸡体内的细胞免疫和体液免疫，具有更强的免疫刺激活性，可 将其开发为免疫增强剂。而壳聚糖季铵盐的季铵基团的取代度是影响其活性的重 要指标之一，但目前没有相关的报道证明多大的取代度能达到最佳的免疫效果。 因此，我们制备了不同取代度的壳聚糖季铵盐，筛选免疫活性最佳的取代度，然 后将该样品制备成负载牛血清白蛋白的纳米粒，研究其免疫活性作用，为完善壳 聚糖季铵盐在免疫增强剂的开发方面提供较全面的理论基础。本文的具体研究结 果如下：

1.成功制备了 6 种不同取代度的含乙酰基壳聚糖季铵盐(HACC)和全脱乙酰 壳聚糖季铵盐(De-HACC)，并以 RAW 264.7 小鼠巨噬细胞为模型，进行免疫活 性的筛选。结果表明，乙酰基显著影响壳聚糖季铵盐的取代度，脱去乙酰基后， 可以得到 71%的高取代度；细胞毒性测试表明在适当浓度范围内所有取代度的壳 聚糖季铵盐均安全无毒；采用 Griess 法测定了一氧化氮(NO)的表达量，荧光定 量 PCR 技术测定了 IL-6 和 TNF-α的 mRNA 表达，发现季铵化程度和乙酰基显著 影响 NO 和细胞因子的表达量，对于不脱乙酰基的壳聚糖季铵化后，30%-HACC 的免疫效果优于 13%-HACC 和 23%-HACC，而全脱乙酰的壳聚糖季铵化后， 54%-De-HACC 的免疫效果优于 62%-De-HACC 和 71%-De-HACC；Caco-2 细胞 不同取代度壳聚糖季铵盐的制备及其对免疫活性的影响 II 对 54%- De-HACC 摄取量高于 30%-HACC。

2. 采 用 离 子 交 联 法 ， 三 聚 磷 酸 钠 为 交 联 剂 ， 分 别 以 30%-HACC 和 54%-De-HACC 为载体包裹牛血清白蛋白，制备了载牛血清白蛋白纳米粒，并对 其进行理化性质表征以及免疫活性测定。结果表明：得到的两种纳米粒粒径为 100 nm 左右，Zeta 电位为+10 mV 左右，具有良好的生物相容性；透射电镜显 示纳米粒成光滑球状结构；聚集性表明 54%-全脱乙酰壳聚糖季铵盐纳米粒 (54%-De-HACC-Nps) 稳 定 性 要 高 于 30%- 含 乙 酰 基 壳 聚 糖 季 铵 盐 纳 米 粒 (30%-HACC-Nps)；且 54%-De-HACC-NPs 对 NO、IL-6 和 TNF-α的表达水平及 被 Caco-2 细胞的摄取均高于 30%-HACC-NPs，说明 54%-De-HACC-NPs 免疫活 性较强。

综上所述，我们证实了取代度在影响壳聚糖季铵盐免疫调节中的重要作用， 为之后壳聚糖季铵盐作为免疫增强剂的开发与应用提供参考。

Chitin, a natural marine biological polysaccharide , whose storage is after only to cellulose and has wide variety of sources. Chitosan is formed by the deacetylation of chitin and has many good biological properties, such as natural non-toxicity, good biocompatibility, antiviral, and immunomodulatory effects. However, its insolubility in water limits its development in the field of medical immunity. The introduction of new active groups into the chitosan chain for modification is one of the effective means to improve the water solubility of chitosan and improve its biological activity. Among the many methods of modification, such as phosphorylation, sulfation and quaternization, the preparation of quaternary ammonium chitosan into a quaternary ammonium salt can effectively improve the water solubility and biological activity, which has attracted much attention. Our research group also conducted partial studies on the immune activity of quaternized chitosan before and the lower molecular weight was found the higher immunological activity. In addition, quaternized chitosan also can activate MAPK. JAK/STAT and PI3K/Akt pathways in RAW264.7 cells to regulate immune response. Futhermore, it was found that preparing the chitosan quaternary ammonium salt into nanoparticles can significantly improve the cellular immunity and humoral immunity in chickens and has strong immunostimulatory activity. However, the degree of substitution of the quaternary ammonium group is one of the important indicators that affect the quaternary ammonium salt of chitosan. Therefore, in order to more completely prove the immunological activity of the quaternary ammonium salt of chitosan and is expected to develop into a powerful immunostimulant, the degree of quaternary ammonium research is essential. Indeed, the research on the degree of quaternary ammonium essential. Therefore, we have prepared quaternary ammonium salts with different quaternary ammonium degrees, and prepared quaternary ammonium salts with better immunological activity into nanoparticles coated with  BSA. The development of immunostimulants provides a more comprehensive theoretical basis. The specific research results of this article are as follows:

1. HACC and De-HACC were successfully prepared with different quaternary ammonium degrees. And the RAW 264.7 mouse macrophages was used as a model for immunological activity teat. Firstly, the MTT method was used to determine the cytotoxicity of cella and then the Griess method was used to determine the expression of NO. It was found that the expression of NO was related to the degree of quaternization. The immune effect of 30%-HACC was better than that of 13%-HACC and 23%-HACC, and 54%-De-HACC has better immune effects than 62%-De-HACC and 71%-De-HACC; the mRNA expression of IL-6 and TNF-α was measured by fluorescence quantitative PCR technology, the results It was found that 30%-HACC induced IL-6 and TNF-α expression levels higher than 13%-HACC and 23%-HACC; 54%- De-HACC induced IL-6 and TNF-α expression levels higher than 62%-De- HACC and 71%-De-HACC; Caco-2 cells uptake 54%-De-HACC higher than 30%-HACC.

2. Sodium tripolyphosphate was used as the cross-linking agent, and the ion cross-linking method was used to coat bovine serum albumin with 30%-HACC and 54%-De-HACC as the carrier, respectively. Then the nanoparticles were characterized to determine physical and chemical properties as well as immunological activity. The average particle size of the nanoparticles was about 100 nm , the zeta potential was abput +10 mV, which attributed to good biocompatibility. Moreover, transmission electron microscopy showed that the nanoparticles were in a smooth spherical structure. The stability of 54%-De-HACC-Nps is higher than that of 30%-HACC-Nps. Moreover, 54%-De-HACC-NPs expressed higher levels of NO, IL-6 and TNF-α than 30%-HACC-NPs, indicating that 54%-De-HACC-NPs has a strong immunological activity.

In summary, we have confirmed the importance of quaternary degree in the RAW264.7cells immune regulation by quaternary chitosant, which provides a powerful reference for the prospective application of chitosan quaternary ammonium salt in immunostimulant