壳聚糖盐酸盐稳定乳液的制备及免疫效果分析

Preparation of chitosan hydrochloride stabilized emulsion and its immunostimulatory effect

摘要:

为了解决油乳佐剂在诱导细胞免疫方面的不足，引入正电荷的壳聚糖盐以稳定乳液，从而提升细胞免疫应答。本研究选取卵清蛋白为模式抗原，分别制备了单抗原疫苗、商品佐剂疫苗和乳液疫苗，表征了乳液的粒径、电势及抗原吸附率等参数。通过肌肉注射免疫BALB/c小鼠，检测免疫后抗体和细胞因子的分泌水平、淋巴细胞的活化水平以评价乳液的免疫效果。结果显示壳聚糖盐酸盐可有效稳定乳液，其粒径在600 nm左右，荷正电并对抗原吸附率达90%以上。免疫动物后，该乳液可有效提升血清特异性抗体水平并增强IL-4的分泌，显著提高抗原特异性CD8⁺T细胞的比例，提升细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL)表面活化分子的表达水平，诱导高效的细胞免疫应答。此外，乳液能够有效提高记忆T细胞(CD44⁺CD62L⁺)的水平。综上所述，以壳聚糖盐酸盐稳定的乳液为佐剂能有效提升体液及细胞免疫效果，并有望起长期保护作用。

Abstract:

In order to increase the ability of oil-emulsion adjuvant to stimulate cellular immunity, chitosan hydrochloride with positive charge was selected to stabilize oil-in-water emulsion (CHE). In this paper, model antigen ovalbumin was selected to prepare vaccines with emulsion adjuvant, commercial adjuvant or no adjuvant. The emulsion was characterized by measuring the particle size, electric potential and antigen adsorption rate. BALB/c mice were immunized by intramuscular injection. Serum antibody levels, the numbers of IL-4-secreting cells in splenocytes, cytotoxic T lymphocyte (CTL) response, and the expression of central memory T cells were measured to evaluate the immunostimulatory effect. The results showed that chitosan hydrochloride can effectively stabilize the emulsion. The emulsion size is about 600 nm, and the antigen adsorption rate is more than 90%. After immunization, CHE could increase serum antibodies levels and increase IL-4 secretion. Expression of CTL surface activation molecules was also increased to stimulate CTL response further and to increase the CD44⁺CD62L⁺ in T cells proportion. CHE as adjuvant can stimulate humoral and cellular immunity more efficiently, and is expected to extend the duration of protection.