通过在小鼠中吸液性淋巴血管阻止淋巴流
1Department of General Surgery, The Second Affiliated Hospital of Harbin Medical University,Harbin Medical University, 2Inflammation Research Network, Department of Microbiology, Immunology and Infectious Diseases, Snyder Institute for Chronic Diseases, Cumming School of Medicine,University of Calgary
Summary
提出了一种通过手术阻断淋巴血管来阻止淋巴流动的方案。
Abstract
淋巴血管通过运输抗原、细胞因子和细胞排干淋巴结（LNs）来保持组织流动性平衡和优化免疫保护至关重要。淋巴流中断是研究淋巴血管功能的重要方法。从杂物足部到流行淋巴结（pLNs）的淋巴血管被明确界定为淋巴排入pLNs的唯一途径。给这些发性淋巴血管进行吸管可以选择性地防止淋巴流向pLAN。此方法允许干扰淋巴流，对排水 pLN 中的淋巴内皮细胞、支淋巴血管以及该区域周围的其他淋巴血管的损害最小。此方法已用于研究淋巴如何影响 LN 中的高内皮淋巴瘤（ HEV ）和化疗表达，以及淋巴如何在没有功能性淋巴血管的情况经 LN 周围的脂肪组织。随着人们越来越认识到淋巴功能的重要性，这种方法将有更广泛的应用，以进一步解开淋巴血管在调节LN微环境和免疫反应的功能。
Introduction
淋巴系统的空间组织提供结构和功能支持，以有效地去除细胞外液体，将抗原和抗原呈现细胞（APC）运送到排出的LNs。最初的淋巴血管（也称为淋巴毛细血管）由于其不连续的细胞间结，便于从周围的细胞外空间1有效地收集液体、细胞和其他材料， 因此具有高度渗透性。最初的淋巴血管合并成收集淋巴血管，这些血管有紧密的细胞间结，连续的地下室膜和淋巴肌肉覆盖。收集淋巴血管负责运输收集的淋巴到排水的LNs，并最终返回淋巴循环2，3。将淋巴推进到排水的LN的收集淋巴血管是发性淋巴血管4，5，6，7。淋巴血管的阻塞可以阻止淋巴流进入LNs，这是研究淋巴流动功能时有用的技术。
先前的研究表明，淋巴流动在运输抗原和药水，以及维持LN平衡方面起着重要作用。众所周知，组织衍生的APCs，通常激活的迁移树突状细胞（DCs），通过发源淋巴血管前往LN激活T细胞8。自由形式的抗原，如微生物或可溶性抗原，被动地与淋巴流动到LN激活LN居民APC的想法在过去十年中得到了接受9，10，11，12。与淋巴一起携带的自由形式抗原在感染后需要几分钟时间才能到达 LN，LN-驻留细胞活化可能在刺激后 20 分钟内发生。这比迁移 DC 的激活要快得多，迁移 DC 进入排空 LN 9 需要 8小时多。除了运输抗原以启动免疫保护外，淋巴还携带细胞因子和DC到LN，以维持其微环境，并支持免疫细胞平衡13，14。此前，通过阻断淋巴血管来阻断淋巴流动表明，淋巴需要维持支持恒流T细胞所需的HV表型，B细胞向LN15、16、17定位。CCL21是一种关键的化学素，它指导直流和T细胞在LN8，18中的定位。阻塞淋巴流会中断 LN 中的 CCL21 表达，并可能中断 LN19中的直流和 T 细胞定位和/或相互作用。因此，阻断淋巴流动可以通过破坏调节 LN 免疫反应的 LN 微环境，直接或间接地消除抗原/直流对排干 LN 的访问。为了更好地研究淋巴流动的功能，提出了一个实验方案（图1），通过将支脚的淋巴血管从脚垫到pLN的吸血，来阻止小鼠的淋巴流动。该方法是今后在健康和患病条件下研究淋巴功能的重要技术。
Protocol
所有动物工作都需要得到机构和政府伦理及动物处理委员会的批准。 这是一个非生存手术。 1. 材料准备 通过将 70 mL 的 100% 乙醇与 30 mL 的无菌水混合，准备 100 mL 的 70% 乙醇。手术前将所有手术工具绝育，并在手术前和手术期间将工具保持为 70% 乙醇，以保持灭菌。 准备注射装置。 切割 ±30 厘米聚乙烯管（直径 0.28 毫米）。将 30 G x 1/2 针（针 A）的尖端?…
Representative Results
淋巴细胞缝合在以前的研究中已经使用15，16，17，19，作为一个重要的工具，研究淋巴流的功能之前，淋巴细胞的分子生物学被更好地理解。阻塞淋巴流中断LN平衡，这导致 HEV 失去最佳淋巴细胞宿主到 LN15、16、 17所需的关键基因表达。<sup…
Discussion
在健康和患病条件下，阻断淋巴流动在操纵抗原输送到LN方面有着广泛的应用。可以使用此方法控制抗原传递的时间，以研究连续淋巴流如何调节排空 LNs 中的免疫反应。这种淋巴流中断方法还可用于研究淋巴如何影响 LN 中的细胞分块、细胞激活、细胞迁移和细胞细胞相互作用。
专门表达人类白喉毒素受体（DTR）的小鼠在淋巴内皮细胞（Flt4-cre-dtr）中已经开发出来;这?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢艾娃·扎迪内扎德对手稿进行校对。这项工作得到了加拿大健康研究所（CIHR，PJT-156035）和加拿大SL创新基金会（32930）以及中国国家自然科学基金会（81901576）的支持。
Materials
| 0.9% Sodium Chloride Saline | Baxter | JB1323 | |
| 100% ethanol | Greenfield Global | University of Calgary distribution services UN1170. | |
| Depilatory cream | Nair | Nair Sensitive Formula Hair Removal Crème with Sweet Almond Oil and Baby Oil, 200-ml. Or similar product. | |
| Evans Blue dye | Sigma Life Science | E2129-10G | For 1 ml of Evans blue dye, add 0.1g Evans blue to 10 ml PBS. The Evens Blue solution will be filtered through 0.22 mm filters and kept sterile in 1ml aliquots. |
| Fluorescein isothiocyanate isomer I (FITC) | Sigma Life Science | F7250-1G | |
| Forceps Dumont #3 | WPI | 500337 | |
| Forceps Dumont #5 | WPI | 500233 | |
| Injection apparatus | Connect one end of polyethylene tubing to 30G × ½ needle. Attach a 1ml TB syringe to the needle. Dislodge needle shaft from another 30G × ½ needle. Insert the blunt end of the 30G × ½ needle shaft into the other end of the tubing. The inside diameter of this tubing is 0.28mm. Thus, 1.6 cm of fluid in the tubing is 1 μl. | ||
| Insulin syringe | Becton Dickinson and Company (BD) | 329461 | |
| IRIS Forcep straight | WPI | 15914 | |
| IRIS scissors | WPI | 14218-G | |
| Ketamine | Narketan | DIN 02374994 | The suppliers of Ketamine and Xylazine are usually under institutional and governmental regulation. |
| Needles (26Gx3/8) | Becton Dickinson and Company (BD) | 305110 | |
| Needles (30Gx1/2) | Becton Dickinson and Company (BD) | 305106 | |
| Paton Needle Holder | ROBOZ | RS6403 | Straight, Without Lock; Serrated |
| Phosphate-Buffered Saline (PBS) | Sigma Life Science | P4417-100TAB | |
| Polyethylene tubing | Becton Dickinson and Company (BD) | 427401 | |
| Surgical tape (1.25cmx9.1m ) | Transpore | 1527-0 | |
| Surgical tape (2.5cmx9.1m ) | Transpore | 1527-1 | |
| Suture | Davis and Geck CYANAMID Canada | 11/04 | 0.7 metric monofilament polypropylene |
| Syringe (1ml) | Becton Dickinson and Company (BD) | 309659 | |
| VANNAS scissors | World Precision Instruments (WPI) | 14122-G | |
| Xylazine | Rompun | DIN02169606 | The suppliers of Ketamine and Xylazine are usually under institutional and governmental regulation. |
| Equipment | |||
| Dissecting microscope | Olympus | Olympus S261 (522-STS OH141791) with light source: Olympus Highlight 3100 | |
| Confocal microscope | Leica | SP8 |
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Cite This Article
Lin, Y., Xue, J., Liao, S. Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice. J. Vis. Exp. (159), e61178, doi:10.3791/61178 (2020).