将IL-33刺激的巨噬细胞过继转移到博来霉素诱导的小鼠模型中，以研究其对体内特发性肺纤维化的影响
Summary
该协议描述了肺间质巨噬细胞（IMs）的分离及其在小鼠模型中IL-33刺激肺泡后的过继转移，这可以促进特发性肺纤维化（IPF）的 体内 研究。
Abstract
早期肺损伤引起的炎症反应是特发性肺纤维化（IPF）发展的重要原因之一，其伴有巨噬细胞和中性粒细胞等炎症细胞的活化，以及包括TNF-α，IL-1β和IL-6在内的炎症因子的释放。已知由活化的肺间质巨噬细胞（IM）响应IL-33刺激引起的早期炎症在IPF的病理过程中起着至关重要的作用。该协议描述了由IL-33刺激的IM的过继转移到小鼠肺部以研究IPF发育。它涉及从宿主小鼠肺部分离和培养原代IM，然后将受刺激的IM过继转移到博来霉素（BLM）诱导的IPF受体小鼠的肺泡中（这些小鼠先前已通过氯膦酸盐脂质体处理耗尽了肺泡巨噬细胞），以及这些小鼠的病理评估。代表性结果表明，IL-33刺激巨噬细胞的过继转移加重了小鼠肺纤维化，表明巨噬细胞过继转移实验的建立是研究IPF病理学的良好技术手段。
Introduction
特发性肺纤维化（IPF）是一种由多种因素引起的弥漫性肺部炎症性疾病1。在Th1和Th2免疫反应的细胞因子微环境中，巨噬细胞可以极化为经典活化的巨噬细胞（M1）和替代活化的巨噬细胞（M2）。脂多糖（LPS）或细胞因子IFN-γ诱导M1巨噬细胞极化并产生促炎细胞因子，包括iNOS，IL-1，IL-6，TNF-α和IL-12。相比之下，II型细胞因子IL-4和IL-13驱动M2巨噬细胞的极化，M2巨噬细胞可以产生不同的成纤维细胞生长促进因子，如TGF-β和PDGF，促进肺纤维化2。IPF的病理过程伴随着巨噬细胞活化和浸润。IPF通过释放细胞因子介导损伤修复、炎症和纤维化3。由于治疗选择有限，探索IPF的分子病理机制对于制定IPF预防和治疗的新策略具有重要意义。我们小组和其他研究人员4，5 先前的研究已经证实IPF患者和博来霉素（BLM）诱导的IPF小鼠模型中IL-33的释放增加。IL-33在纤维化过程中由上皮和内皮细胞释放并参与巨噬细胞活化，导致成纤维细胞异常增殖，白细胞浸润，最终丧失肺功能5。目前的协议描述了IL-33刺激的间质巨噬细胞（IMs）向肺泡的过继转移，作为研究小鼠模型中IPF发育的一种手段。在这里，从宿主小鼠的肺组织中分离IMs， 体外培养，用IL-33刺激24小时，然后通过气管注射过继转移到受体小鼠的肺泡中。与以前的研究相比，直接收集受刺激的小鼠巨噬细胞并将其过继转移到受体肺泡中会加重肺纤维化的程度，并且可以更清楚地说明刺激因子对纤维化的影响6。本文中描述的技术可以使研究人员探索由潜在细胞因子刺激的巨噬细胞在IPF发展中的功能。
Protocol
Representative Results
Discussion
本研究为去除、分离、培养和转移巨噬细胞提供了一种有效的方法，有助于研究小鼠肺纤维化的机制。小鼠巨噬细胞耗竭的方法很多，如气管给药、尾静脉注射和鼻吸11。本研究优化了鼻吸法，操作简单，可有效耗竭肺巨噬细胞8，9。在IL-33刺激培养中的IMs24小时后，使用非侵入性气管给药途径将IM过继转移到受体小鼠的肺部，这对小鼠?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者认可江南大学实验室管理专题：基于病理标本的数字切片库构建（JDSYS202223）和国家自然科学基金（81800065）。
Materials
| DMEM | Life technologies Biotechnology,USA | 1508012 | |
| Arterial indwelling needle | B Braun Melsingen AG,Germany | 21G15G8393 | |
| BD Accuri C6 Plus | Becton Dickinson,USA | ||
| Bleomycin | Biotang, USA | Ab9465 | |
| Carbon dioxide incubator | Thermo Forma, USA | Thermo Forma370 | |
| CD11b | R&D Systems,USA | 1124F | |
| CD11c | R&D Systems,USA | N418 | |
| Cell culture dish | Thermo Forma, USA | 174926 | |
| Clodronate liposomes | Clodronate liposomes,Netherlands | CI-150-150 | |
| Collagenase A | Sigma-Aldrich, USA | 10103578001 | |
| F4/80 | R&D Systems,USA | 521204 | |
| Falcon Cell Strainer | Becton,Dickinson and Company, USA | 352340 | |
| Fetal bovine serum (FBS) | Life technologies,USA | 1047571 | |
| Hematoxylin Eosin | Nanjing Jiancheng Technology,China | 06-570 | |
| LightCycler 480 PCR detection system | Roche, USA | ||
| Murine recombinant factor IL-33 | Peprotech, USA | 210-33 | |
| Nikon microscope | Nikon Corporation, Japan | 941185 | |
| Penicillin, streptomycin | Life technologies,USA | 877113 | |
| Phosphate buffer (PBS) | Guangdong Huankai Microbial Technology ,China | 1535882 | |
| RBC lysis buffer | Beyotime Biotechnology Company,China | C3702 | |
| RNA Isolater | Vazyme company,China | R401-01-AA | Total RNA extraction reagent |
| RWD Inhalation Anesthesia Machine | Shenzhen Rayward Life Technology ,China | R500 | |
| Semi-automatic paraffin slicer | Leica, Germany | LeicaRM2245 | |
| SYBR Premix Ex Taq | Takara, Japan | 410800 | |
| Trypsin 0.25% | Life Technologies, USA | 1627172 |
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