体外 纳米乳剂疫苗佐剂蛇腓磷素D的细胞活性评价
Summary
该协议提供了评估纳米乳剂蛇腓苷D佐剂是否促进有效细胞免疫应答的详细方法。
Abstract
作为疫苗的主要成分，佐剂可以直接诱导或增强与抗原相关的强大、广泛、先天和适应性免疫应答。Ophiopogonin D（OP-D）是从植物 Ophiopogon japonicus中提取的纯化成分，已被发现可用作疫苗佐剂。采用低能乳化法制备纳米乳液蛇腓磷素D（NOD）可以有效克服OP-D溶解度低、毒性低的问题。在本文中，研究了一系列用于细胞活性评估的 体外 方案。 使用细胞计数试剂盒-8测定法测定L929的细胞毒性作用。然后，采用ELISA和ELISpot方法检测免疫小鼠脾细胞刺激培养后分泌的细胞因子水平和相应的免疫细胞数。此外，通过激光扫描共聚焦显微镜（CLSM）观察从C57BL / 6小鼠中分离并在与GM-CSF加IL-4孵育后成熟的骨髓来源树突状细胞（BMDCs）中的抗原摄取能力。重要的是，在用佐剂共培养空白小鼠的腹膜巨噬细胞（PM）24小时后，通过ELISA试剂盒测量IL-1β，IL-6和肿瘤坏死因子α（TNF-α）细胞因子的水平来确认巨噬细胞活化。希望该协议将为其他研究人员提供直接有效的实验方法来评估新型疫苗佐剂的细胞反应有效性。
Introduction
疫苗是预防和治疗传染病和非传染性疾病的重要手段。在疫苗制剂中适当添加佐剂和递送载体有利于增强抗原的免疫原性并产生持久的免疫应答1。除了经典的佐剂明矾（铝盐）外，目前上市的疫苗佐剂还有六种：MF592，3，AS04 3，AS03 3，AS01 3，CpG10184和Matrix-M5。一般来说，当人体遇到病毒攻击时，第一道和第二级防线（皮肤、粘膜、巨噬细胞）率先清除病毒，最后激活涉及免疫器官和免疫细胞的第三道防线。自 1920 年代初以来，铝盐和铝盐一直是人类疫苗中使用最广泛的佐剂，可引发有效的先天免疫反应6。然而，有人提出，经典佐剂激活抗原呈递细胞（APC），刺激免疫细胞产生特定的细胞因子和趋化因子，是佐剂起作用的机制，可能是佐剂仅对特异性免疫应答产生瞬时作用的原因之一7。有限的人用许可佐剂的存在是开发引起有效免疫反应的疫苗的限制因素8。
目前，越来越多的佐剂研究正在证明在小鼠中诱导强烈细胞免疫反应的能力。QS-21已被证明可诱导平衡的T辅助T-1（Th1）和T辅助2（Th2）免疫应答，产生更高水平的抗体滴度，并延长作为佐剂的保护，但其强毒性和溶血特性限制了其作为独立临床佐剂的发展9，10。OP-D（芦荟皂苷元-O-α-L-鼠李糖吡喃糖1-（1→2）-β-D-吡喃木糖基-（1→3）-β-D-呋喃糖苷）是从中药植物甾本科植物甾体皂苷4中分离出的甾体皂苷之一。此外，它是在黄桐中发现的主要药理活性成分（神麦山），已知具有一定的药理特性11。此外，它是百合科的成员，因其在细胞炎症和心肌损伤中的抑制和保护作用而被广泛使用。例如，OP-D改善了BALB / c小鼠中DNCB诱导的特应性皮炎样病变和肿瘤坏死因子α（TNF-α）炎性HaCaT细胞12。重要的是，OP-D通过减少活性氧的产生和破坏线粒体膜损伤，促进心血管系统的抗氧化保护，并保护心脏免受阿霉素诱导的自噬损伤。实验表明，服用OP-D和单桥粒苷有助于增强免疫健康，增加白细胞计数和DNA合成，并使抗体持续更长时间13。先前已发现OP-D具有佐剂作用14。
纳米乳液是由表面活性剂、油、助表面活性剂和水的组合组成的水包油纳米制剂12，15。这些纳米疫苗设计允许将抗原和佐剂封装在一起，以增强免疫刺激，保护抗原并促进树突状细胞（DC）成熟16。为了开发从筛选中获得的这些新型佐剂，重要的是找到适当的方法来评估它们的细胞反应能力。
该协议的目的是系统评估佐剂是否可以增强 体外 细胞培养中的吞噬作用和免疫细胞的表达，并详细说明主要的实验方法。实验分为四个小节：（1）OP-D和NOD对L929细胞的毒性通过细胞计数试剂盒-8（CCK-8）测定法确定;（2）通过脾细胞刺激和ELISpot测定检测免疫小鼠内分泌IFN-γ和IL-17A的细胞因子水平以及相应的细胞数;（3）使用共聚焦显微镜观察辅助刺激后DC的抗原呈递能力;（4）检测与佐剂共培养的正常小鼠腹膜巨噬细胞（PMs）获得的上清液中的三种细胞因子IL-1β，IL-6和TNF-α。
Protocol
Representative Results
Discussion
亚单位疫苗具有极佳的安全性，但免疫原性较差。增强免疫原性的主要策略是物理吸附抗原或偶联佐剂，并将其纳入药物递送系统，以促进DC的摄取和呈递。 天然植物皂苷如奎莱皂苷及其衍生物剧毒，不适合开发人类疫苗17。因此，研究疫苗或佐剂对细胞的毒性作用是评价新疫苗的必要第一步。
本研究中提出的方案是根据ISO 10993-5：200918进?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究获得国家重点研发计划2021YFC2302603号资助，国家自然科学基金项目第31670938、32070924、82041045、32000651号，重庆市自然科学基金项目第2014jcyjA0107号和2019jcyjA-msxmx0159号资助，北京市研究生科研与创新项目CYS21519号资助， 陆军军医大学专项拨款2020XBK24号，全国大学生创新创业计划202090031021号资助。
Materials
| 0.25% Trypsin-EDTA (1x) | GIBCO, USA | 25200056 | |
| 96-well filter plates | Millipore. Billerica, MA | CLS3922 | |
| AlPO4 | General Chemical Company, USA | null | |
| Automated Cell Counter | Countstar, China | IC1000 | |
| BALB/c mice and C57BL/6 mice | Beijing HFK Bioscience Co. Ltd | null | |
| caprylic/capric triglyceride (GTCC) | Beijing Fengli Pharmaceutical Co. Ltd., Beijing, China | null | |
| CCK-8 kits | Dojindo, Japan | CK04 | |
| Cell Counting Plate | Costar, Corning, USA | CO010101 | |
| Cell Sieve | biosharp, China | BS-70-CS | |
| Centrifuge 5810 R | Eppendorf, Germany | 5811000398 | |
| DAPI | Sigma-Aldrich, St. Louis, USA | D9542 | |
| DMEM basic(1x) medium | GIBCO, USA | C11885500BT | |
| DSZ5000X Inverted Microscope | Nikon,Japan | DSZ5000X | |
| EL-35 (Cremophor-35) | Mumbai, India | null | |
| ELISpot classic | AID, Germany | ELR06 | |
| Fetal Bovine Serum | GIBCO, USA | 10099141C | |
| Full-function Microplate Reader | Thermo Fisher Scientific, USA | VL0000D2 | |
| GFP | Sigma-Aldrich, St. Louis, USA | P42212 | |
| Glutamax | Invitrogen, USA | 35050061 | |
| Granulocyte Macrophage Colony-Stimulating Factor | GM-CSF, R&D Systems, USA | 315-03 | |
| HEPES | Invitrogen, USA | 15630106 | |
| HF 90/240 Incubator | Heal Force, Switzerland | null | |
| IL-4 | PeproTech, USA | 042149 | |
| L929 cell line | FENGHUISHENGWU, China | NCTC clone 929 (RRID:CVCL_0462) | |
| Laser Scanning Confocal Microscopy | Zeiss, Germany | LSM 980 | |
| MONTANE 85 PPI | SEPPIC, France | L12910 | |
| MONTANOX 80 PPI | SEPPIC, France | 36372K | |
| Mouse IFN-γ ELISA kit | Dakewe, China | 1210002 | |
| Mouse IFN-γ precoated ELISPOT kit | Dakewe, China | DKW22-2000-096 | |
| Mouse IL-17A ELISA kit | Dakewe, China | 1211702 | |
| Mouse IL-17A ELISpotPLUS Kit | ebiosciences, USA | 3521-4HPW-2 | |
| Mouse IL-1β ELISA kit | Dakewe, China | 1210122 | |
| Mouse IL-6 ELISA kit | Dakewe, China | 1210602 | |
| Mouse TNF-α ELISA kit | Dakewe, China | 1217202 | |
| Non-essential amino acids(100x) | Invitrogen, USA | 11140050 | |
| Ophiopogonin-D | Chengdu Purui Technology Co. Ltd | 945619-74-9 | |
| Penicillin-Streptomycin Solution | Invitrogen, USA | 15070063 | |
| Phalloidin | Solarbio, China | CA1620 | |
| Phosphate Buffered Saline | ZSGB-BIO, China | ZLI-9062 | |
| Red Blood Cell Lysis Buffer | Solarbio, China | R1010 | |
| RPMI 1640 medium | Hyclone (Life Technology), USA | SH30809.01 | |
| Sodium pyruvate(100 mM) | Invitrogen, USA | 11360070 | |
| Squalene | Sigma, USA | S3626 | |
| β- Mercaptoethanol | Invitrogen, USA | 21985023 |
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