白术多糖超大介孔二氧化硅纳米颗粒的制备及其黏膜免疫佐剂活性的研究

doi:10.11843/j.issn.0366-6964.2025.05.045

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (5): 2507-2519.doi: 10.11843/j.issn.0366-6964.2025.05.045

白术多糖超大介孔二氧化硅纳米颗粒的制备及其黏膜免疫佐剂活性的研究

林欣怡¹(), 蒋欣雨¹, 苏子诺¹, 王煜灵¹, 阮诗雨¹, 洪海龙¹, 吴佳豪¹, 伯若楠¹^,²^,*()

1. 扬州大学兽医学院, 扬州 225009
2. 江苏省高校动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009

收稿日期:2024-05-08 出版日期:2025-05-23 发布日期:2025-05-27
通讯作者: 伯若楠 E-mail:mzdzh@foxmail.com;007309@yzu.edu.cn
作者简介:林欣怡(2003-)，女，江苏南京人，本科生，主要从事中药成分新型纳米制剂免疫研究，E-mail：mzdzh@foxmail.com
基金资助:
国家自然科学基金(32002324)；江苏省自然科学基金(BK2020945)；江苏省高校优势学科建设工程资助项目(JS20130601)；扬州大学大学生创新创业训练计划项目(XCX20230741)；扬州大学教学改革研究课题(YZUJX2023-C13)

Study on Preparation and the Mucosal Immune Adjuvant Activity of Ultra-Large Mesoporous Silica Nanoparticles Loading Polysaccharide from Atractylodes macrocephala Koidz

LIN Xinyi¹(), JIANG Xinyu¹, SU Zinuo¹, WANG Yuling¹, RUAN Shiyu¹, HONG Hailong¹, WU Jiahao¹, BO Ruonan¹^,²^,*()

1. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China
2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China

Received:2024-05-08 Online:2025-05-23 Published:2025-05-27
Contact: BO Ruonan E-mail:mzdzh@foxmail.com;007309@yzu.edu.cn

摘要/Abstract

摘要：

旨在结合白术多糖和超大介孔二氧化硅两者的优势，开发新型中药多糖黏膜免疫佐剂。本研究首先制备白术多糖超大介孔二氧化硅(AMP-UCMS)纳米颗粒并对其进行一系列表征，包括透射电镜、粒径、电势、聚合物分散性指数(polymer dispersity index，PDI)和红外光谱，然后进一步研究口服AMP-UCMS作为H9N2型禽流感灭活病毒的黏膜免疫佐剂活性。试验选取150只雏鸡随机分成6组并进行不同免疫处理，分别为阴性对照(Control)、全灭活病毒(whole inactivated virus, WIV)、WIV+AMP、WIV+UCMS、WIV+AMP-UCMS、市售疫苗(Vaccine)，其中各组选取6只雏鸡用H9N2禽流感病毒滴鼻攻毒。通过血凝抑制(HI)试验测定各组鸡血清HI抗体效价；采用ELISA方法检测空肠H9N2型禽流感病毒特异性sIgA抗体水平；利用HE染色观察空肠和肺的组织形态变化；利用实时荧光定量PCR(RT-qPCR)测定空肠IgA J-chain、pIgR、TNF-α和IL-1β的mRNA表达水平以及攻毒后鸡的肺组织病毒载量等各项指标。试验结果显示，与Control组相比，WIV+AMP-UCMS组可极显著提高鸡血清HI抗体效价(P < 0.000 1)和空肠特异性sIgA抗体水平(P < 0.01)，并能够极显著降低肺组织病毒载量(P < 0.000 1)，鸡空肠组织中IgA J-chain(P < 0.001)、pIgR(P < 0.000 1)、TNF-α和IL-1β(P < 0.000 1)的mRNA表达水平都极显著提高，此外还能够增加鸡空肠iIEL数量，降低攻毒后肺组织的损伤。综上表明，白术多糖被成功装载到超大介孔二氧化硅中，AMP-UCMS能够有效诱导鸡的全身免疫和黏膜免疫应答。这些研究结果为新型中药多糖黏膜免疫佐剂在畜禽口服疫苗领域的开发应用提供了参考。

关键词: 白术多糖, 超大介孔二氧化硅, 黏膜免疫佐剂, 表征, H9N2灭活病毒

Abstract:

The aim of this study was to develop a new type of traditional Chinese medicine polysaccharide mucosal immune adjuvant by combining the advantages of polysaccharide from Atractylodes macrocephala Koids and ultra-large mesoporous silica. First, ultra-large mesoporous silica nanoparticles loading polysaccharide from Atractylodes macrocephala Koidz (AMP-UCMS) was prepared and a series of characterizations were carried out on it, including transmission electron microscopy, particle size, potential, polymer dispersity index (PDI) and infrared spectroscopy. Then, the mucosal immune adjuvant activity of oral AMP-UCMS as an inactivated H9N2 avian influenza virus was further studied. In the experiment, 150 chicks were randomly divided into 6 groups and received different immunization treatments, namely negative control (Control), whole inactivated virus (WIV), WIV+AMP, WIV+UCMS, WIV+AMP-UCMS, and commercially available vaccine (Vaccine). Among them, 6 chicks from each group were intranasally challenged with H9N2 avian influenza virus. The hemagglutination inhibition (HI) test was used to determine the HI antibody titer of chicken serum in each group; the ELISA method was used to detect the specific sIgA antibody level of H9N2 avian influenza virus in the jejunum; the HE staining was used to observed the histomorphology changes of the jejunum and lung; real-time fluorescent quantitative PCR (RT-qPCR) was used to determine the mRNA expression levels of IgA J-chain, pIgR, TNF-α and IL-1β in the jejunum and various indicators such as the viral load in the lungs of chickens after challenge. The results showed that compared with the Control group, WIV+AMP-UCMS group could highly significantly increase the HI antibody titer of chicken serum (P < 0.000 1) and the specific sIgA antibody level in the jejunum (P < 0.01), and highly significantly reduce the viral load in the lungs (P < 0.000 1), and the mRNA expression levels of chicken jejunum IgA J-chain(P < 0.001), pIgR(P < 0.000 1), TNF-α and IL-1β (P < 0.000 1) were all highly significantly increased. Additionally, it could increase the number of intraepithelial lymphocytes (iIELs) in the chicken jejunum and reduce lung tissue damage following viral challenge. In conclusion, polysaccharide from Atractylodes macrocephala Koidz was successfully loaded into ultra-large mesoporous silica in the current study, and AMP-UCMS could effectively induce systemic immunity and mucosal immune responses in chickens. These research results provide a reference for the development and application of new traditional Chinese medicine polysaccharide mucosal immune adjuvants in the field of oral vaccines for livestock and poultry.

Key words: polysaccharide from Atractylodes macrocephala Koidz, ultra-large pore mesoporous silica nanoparticles, mucosal immune adjuvants, characterization, inactivated H9N2 avian influenza virus

中图分类号:

S859.797

林欣怡, 蒋欣雨, 苏子诺, 王煜灵, 阮诗雨, 洪海龙, 吴佳豪, 伯若楠. 白术多糖超大介孔二氧化硅纳米颗粒的制备及其黏膜免疫佐剂活性的研究[J]. 畜牧兽医学报, 2025, 56(5): 2507-2519.

LIN Xinyi, JIANG Xinyu, SU Zinuo, WANG Yuling, RUAN Shiyu, HONG Hailong, WU Jiahao, BO Ruonan. Study on Preparation and the Mucosal Immune Adjuvant Activity of Ultra-Large Mesoporous Silica Nanoparticles Loading Polysaccharide from Atractylodes macrocephala Koidz[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2507-2519.

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分组Group	处理方式Treatment
Control	口服0.3 mL生理盐水
WIV	口服0.3 mL WIV
WIV+AMP	口服12 mg AMP和0.3 mL WIV
WIV+UCMS	口服12 mg UCMS和0.3 mL WIV
WIV+AMP-UCMS	口服12 mg AMP-UCMS和0.3 mL WIV
Vaccine	肌注0.3 mL市售H9N2灭活疫苗

基因名称 Gene Name	引物序列(5′→3′) Primer sequences (5′→3′)
pIgR-F	TCGATGTCAGCCTGGAGGTC
pIgR-R	AGGGCATTCAATGGTCACATTTC
IgA J-chain-F	GGATCCTAATGAGGACATTGTGGAG
IgA J-chain-R	CTGGGTGGCAGTAACAACCTGA
H9N2-M-F	TGAGCGTGAACACAAACCCT
H9N2-M-R	TGAGCGTGAACACAAACCCA
IL-1β-F	ATCTCGCAGCAGCACATCA
IL-1β-R	CCAGCAGGTTATCATCATCATCC
TNF-α-F	ACTCCAGGCGGTGCCTATGT
TNF-α-R	GTGAGGGTCTGGGCCATAGAA
GADPH-F	ATGGTGAAGGTCGGTGTGAA
GADPH-R	CCTTGACTGTGCCGTTGAAT

组分 Component	体积/μL Volume
Hieff^® qPCR SYBR Green Master Mix	10
正向引物Forward Primer (10 μmol·L^－1)	0.4
反向引物Reverse Primer (10μmol·L^－1)	0.4
模板DNA Template DNA	6
无菌超纯水Ultrapure Water	3.2

程序 Process	温度 Temperature	时间 Time	循环数 Cycle number
预变性Pre-denaturation	95℃	5 min	1
变性Denaturation	95℃	10 s	40
退火、延伸Refolding, extend	60℃	30 s	40
熔解曲线Melting curve	仪器默认设置 Instrument default setting		1

样品Sample	UCMS	AMP-UCMS
粒径/nm Particle size	390.4±9.19	523.0±6.12
聚合物分散性指数Polymer dispersity index (PDI)	0.243±0.01	0.323±0.03
Zeta电位/mV Zeta potential	-8.23±0.26	-17.4±0.30

白术多糖超大介孔二氧化硅纳米颗粒的制备及其黏膜免疫佐剂活性的研究

Study on Preparation and the Mucosal Immune Adjuvant Activity of Ultra-Large Mesoporous Silica Nanoparticles Loading Polysaccharide from Atractylodes macrocephala Koidz

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