从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案
概要
在这里，我们提出了一种经济有效的方法，在用10 ng / mL GM-CSF / IL-4培养7天后从小鼠中分离和产生高纯度骨髓来源的树突状细胞。
Abstract
随着免疫学研究的进展，对树突状细胞（DC）的需求正在逐渐增加。然而，DC在所有组织中都很少见。分离DC的传统方法主要涉及通过注射大剂量（>10 ng / mL）的粒细胞 – 巨噬细胞集落刺激因子/ 白细胞介素-4（GM-CSF / IL-4）来诱导骨髓（BM）分化为DC，使该过程复杂且昂贵。在该方案中，使用在10ng / mL GM-CSF / IL-4培养基中培养的所有BM细胞，经过3-4次半培养交换后，每只小鼠（两个股骨）收获高达2.7 x 107 CD11c + 细胞（DC），纯度为80%-95%。培养10天后，CD11c，CD80和MHC II的表达增加，而细胞数量减少。培养7天后细胞数量达到峰值。此外，该方法仅需10分钟即可收获所有骨髓细胞，并且在培养1周后获得大量DC。
Introduction
树突状细胞（DC）是最强大的抗原呈递细胞（APC），用于激活幼稚的T细胞并诱导针对传染病，过敏性疾病和肿瘤细胞的特定细胞毒性T淋巴细胞（CTL）反应1，2，3。DC是先天免疫和适应性免疫之间的主要纽带，在免疫防御和维持免疫耐受性方面起着至关重要的作用。在过去的40年中，许多研究人员试图定义DC的亚群及其在炎症和免疫中的功能。根据这些研究，DC从骨髓细胞沿着骨髓和淋巴系发展。近年来，肿瘤疫苗取得了重大的里程碑，并拥有光明的未来。在机械上，肿瘤疫苗通过使用肿瘤抗原激活细胞毒性T淋巴细胞来调节免疫反应并防止肿瘤生长。基于DC的疫苗在肿瘤免疫治疗中起着重要作用，已被确定为最有前途的抗肿瘤疗法之一1，4。此外，DC已广泛应用于新型分子靶向药物和免疫检查点抑制剂的检测5.
研究人员迫切需要大量的高纯度DC来进一步研究DC的作用。然而，DC在各种组织和血液中很少见，仅占人类和动物血细胞的1%。骨髓树突状细胞（BMDC）的 体外 培养是获得大量DC细胞的重要方法。同时，用于从骨髓中生成DC的Lutz协议已被研究人员广泛使用6。虽然该方案在获得DC细胞方面是有效的，但它是复杂和昂贵的，涉及添加高浓度的细胞因子和红细胞的裂解。
在这项研究中，我们报告了一种从小鼠骨髓（BM）中分离几乎所有骨髓细胞的方法，并在 体外孵育7-9天后诱导分化为BMDC，GM-CSF和IL-4的浓度较低。该过程只需10分钟即可收获几乎所有骨髓细胞并将其悬浮在完整的培养基中。简而言之，我们在本研究中为BMDC提供了一种高效且具有成本效益的养殖方法。
Protocol
Representative Results
Discussion
人类和小鼠具有不同的DC亚群，包括经典DC（cDC，包括cDC1和cDC2s），浆细胞样DC（pDC）和单核细胞衍生DC（MoDC）9，10，11。人们普遍认为，cDC1s调节细胞毒性T淋巴细胞（CTL）对细胞内病原体和癌症的反应，cDC2调节对细胞外病原体，寄生虫和过敏原的免疫反应12。在GM-CSF和IL-46，<sup class="…
開示
The authors have nothing to disclose.
Acknowledgements
本研究由天津市科技计划（20JCQNJC00550）、天津市卫生科技计划（TJWJ202021QN033和TJWJ202021QN034）资助。
Materials
| β-Mercaptoethanol | Solarbio | M8211 | |
| 6-well plate | Corning | 3516 | |
| APC-MHC II | Biolegend | 116417 | |
| FBS | Gibco | 10100 | |
| PE-CD80 | Biolegend | 104707 | |
| Penicillin-Streptomycin | Solarbio | P1400 | |
| Percp/cy5.5-CD11c | Biolegend | 117327 | |
| PRMI-1640 | Thermo | 11875093 | |
| Recombinant Mouse GM-CSF | Solarbio | P00184 | |
| Recombinant Mouse IL-4 | Solarbio | P00196 | |
| TruStain Fc PLUS (anti-mouse CD16/32) Antibody | Biolegend | 156603 |
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