共载丹参多糖和Mn2+的PLGA纳米粒作为灭活PCV2的佐剂效果评价

doi:10.11843/j.issn.0366-6964.2025.08.040

摘要/Abstract

摘要：

本研究利用PLGA包封了免疫刺激效果强和生物安全性良好的丹参多糖和Mn²⁺，评价了该体系作为灭活PCV2佐剂的免疫增强效果。首先我们利用复乳溶剂挥发法制备了共载丹参多糖和Mn²⁺的PLGA纳米佐剂递送系统(SM-PLGA)，将SM-PLGA与PCV2抗原混合，对小鼠左右大腿皮下分别注射100 μL进行初免，同时设立空白对照组、PCV2组、Alhydrogel (Algel)-PCV2组、丹参多糖-PLGA组、SM组、丹参多糖组和PLGA组，免疫后的第14天对小鼠进行加强免疫。初免后的第5天通过流式细胞术评估腘淋巴结和腹股沟淋巴结中DCs的活化情况。在初免后的第21、28、35、42天收集小鼠血清，对PCV2特异性抗体IgG、IgG1、IgG2a进行动态监测，评价佐剂对PCV2特异性体液免疫反应的增强效果。在初免后的第21、28天采集小鼠脾脏淋巴细胞，分别对CD4⁺T细胞、CD8⁺T细胞、细胞毒性T细胞、记忆性T细胞和生发中心B细胞活化情况进行测定，并对第28天细胞上清中IFN-β、IFN-γ、IL-6进行测定。最后，在初免后的第35天采集小鼠淋巴结，测定生发中心B细胞的活化情况。通过上述不同时段的测定结果，评价SM-PLGA对机体免疫反应的增强效果。由结果可知SM-PLGA在初免后的第5天可以极显著促进淋巴结DCs的活化，在初免后第21~42天可以持续刺激PCV2特异性抗体IgG及其亚型的分泌。在初免后第21天SM-PLGA可显著促进脾脏CD4⁺T细胞、CD8⁺T细胞和CD8⁺T细胞中CTL应答，并在初免后第28和35天仍对记忆性T细胞和生发中心B细胞有较强的刺激作用，同时极显著促进相关细胞因子IFN-β、IFN-γ、IL-6的分泌。结果表明SM-PLGA作为灭活PCV2的佐剂，可以诱导强效的Th1/Th2免疫应答，为PCV2的预防提供参考。

关键词: 丹参多糖, Mn²⁺, PLGA, 佐剂, 细胞免疫, 体液免疫

Abstract:

In this study, PLGA was used to encapsulate Salvia miltiorrhiza polysaccharide and Mn²⁺ with potent immune stimulation effect and good biosafety, and the immune enhancement effect of the system as an inactivated PCV2 adjuvant was evaluated. Firstly, we prepared a PLGA nano-adjuvant delivery system (SM-PLGA) co-loaded with Salvia miltiorrhiza polysaccharide and Mn²⁺ by double emulsion solvent evaporation method. The SM-PLGA was mixed with PCV2 antigen, and 100 μL was injected subcutaneously respectively into the left and right thighs of mice for the first immunization. At the same time, PCV2 group, Alhydrogel (Algel)-PCV2 group and the Control group were established, and on the 14th day after the first immunization, mice were immunized twice. Then, the activation of DCs in popliteal lymph nodes and inguinal lymph nodes were evaluated by flow cytometry on the 5th day after the first immunization. The serum of mice was collected on the 21st, 28th, 35th and 42nd day after the first immunization, and the PCV2 specific antibodies IgG, IgG1 and IgG2a were dynamically monitored to evaluate the enhancement effect of the adjuvant on PCV2 specific humoral immune response. The spleen lymphocytes of mice were collected on the 21st and 28th day after the first immunization, and the activation of CD4⁺T cells, CD8⁺T cells, CTL and memory T cells were measured, and the IFN-β, IFN-γ and IL-6 in the supernatant of the cells on the 28th day were measured. Finally, the lymph nodes of mice were collected on the 35th day after the first immunization to determine the activation of germinal center B cells. Through the results of the above three periods, the enhancement effect of SM-PLGA on cellular immune response was evaluated. SM-PLGA could significantly promote the activation of lymph node DCs on the 5th day after the first immunization, and continuously stimulate the secretion of PCV2-specific antibody IgG and its subtypes on the 21st to 42nd day after the first immunization. SM-PLGA could significantly promote the spleen CD4⁺T cells, CD8⁺T cells and CTL response on the 21st day after the first immunization, and still had a strong stimulating effect on memory T cells and Germinal center B cells on the 28th and 35th day after the first immunization. At the same time, it significantly promoted the secretion of related cytokines, IFN-β, IFN-γ and IL-6. The results showed that SM-PLGA, as an adjuvant for inactivating PCV2, could induce a potent Th1/Th2 immune response and provide a reference for the prevention of PCV2.

Key words: Salvia miltiorrhiza polysaccharide, Mn²⁺, PLGA, adjuvant, cellular immunity, humoral immunity

中图分类号:

S852.65

朱怡萱, 谷鹏飞, 赵奇, 徐盼盼, 范迎赛, 包永占, 王霄, 史万玉. 共载丹参多糖和Mn²⁺的PLGA纳米粒作为灭活PCV2的佐剂效果评价[J]. 畜牧兽医学报, 2025, 56(8): 4031-4041.

ZHU Yixuan, GU Pengfei, ZHAO Qi, XU Panpan, FAN Yingsai, BAO Yongzhan, WANG Xiao, SHI Wanyu. Evaluation of PLGA Nanoparticles Co-loaded with Salvia miltiorrhiza Polysaccharide and Mn²⁺ as an Adjuvant for Inactivating PCV2[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 4031-4041.

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时间 Time	目标细胞 Target cells	染色抗体^[29] Staining antibody	细胞因子 Cytokine
第21天 Day 21	CD3⁺CD4⁺; CD3⁺CD8a⁺	CD3-FITC; CD4-APC; CD8a-PE	颗粒酶B
	细胞毒性T细胞	CD8a-PE; CD107a(lamp-1)-FITC; CD178 FASL-APC
第28天 Day 28	记忆T细胞	CD4-APC；CD8a-PE；CD44-PE-Cyanine7；CD62L(L-Selectin)-FITC；	IFN-γ IFN-β IL-6

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共载丹参多糖和Mn²⁺的PLGA纳米粒作为灭活PCV2的佐剂效果评价

Evaluation of PLGA Nanoparticles Co-loaded with Salvia miltiorrhiza Polysaccharide and Mn²⁺ as an Adjuvant for Inactivating PCV2

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