华西医学

华西医学

小鼠肺部微循环活体显微成像

查看全文

目的鉴于肺部疾病的高发病率以及活体动物实验在揭示生物现象方面的优势,建立一套利用共聚焦显微镜进行小鼠肺部活体原位成像的方法。方法将 10 只 BALB/c 雄性小鼠随机分为 A、B 两组,每组各 5 只。利用自制的稳定装置,控制小鼠局部肺组织的运动以获取稳定的观察面,然后用激光共聚焦显微镜进行视频拍摄(5 min)。拍摄过程中,A 组小鼠经尾静脉注射异硫氰酸荧光素-葡聚糖,B 组注射绿色荧光蛋白-血小板(经 tie2-cre&rosa26-tomato-EGFP 转基因 black C57 雄性小鼠的血液提取),A 组用于观察肺部灌注情况以及该方法对肺组织的损伤情况,B 组用于观察血小板运动的情况。结果通过该套方法获得了肺组织结构清晰、稳定的图像,随时间推移,5 只小鼠第 0、30、60、120、180 和 300 秒这 6 个时间点肺泡面积的均值依次为(1 603±181)、(1 588±183)、(1 528±363)、(1 506±353)、(1 437±369)、(1 549±307) μm2,肺泡大小未随时间变化(P>0.05);视频画面流畅,血小板的快速运动均被记录而且颗粒清晰、无拖尾;观察结束后,苏木精-伊红染色显示肺组织无明显损伤。结论该套方法可用于小鼠肺部活体状态下微循环系统的观察和研究,为其他肺部疾病在活体状态下的研究提供了方法学基础。

ObjectiveTo design a method for observing pulmonary microcirculation in vivo in a native tissue environment, due to the high incidence of lung diseases and the advantages of animal experiments in vivo.MethodsTen BALB/c male mice were randomly divided into group A and group B, with five mice in each group. A self-made apparatus was used to control the movement towards local lung tissues in order to get a stabilized observation plane, and then a 5-minute video was shot with laser confocal scanning microscope. During the filming, the mice in group A were injected with fluorescein isothiocyanate-dextran via the tail vein, and the mice in group B were injected with green fluorescent protein-platelets (extracted from the blood of tie2-cre&rosa26-tomato-EGFP transgenic black C57 male mice). The data of group A was used to observe the lungs perfusion and the damage to tissue by this method, and the data of group B was used to observe the movement of platelets.ResultsImage of lung structure obtained by this method was clear and stable. Mean areas of alveolus in an instant and at the 30th, 60th, 120th, 180th, and 300th second were (1 603±181), (1 588±183), (1 528±363), (1 506±353), (1 437±369), (1 549±307) μm2, respectively, and there were no significant differences between each time point (P>0.05). The video was smooth, the rapid movement of platelets was recorded and the particles were clear and without tailing; after the observation, hematoxylin-eosin staining showed no obvious damage to the lung tissue.ConclusionThe method can be used for the observation and research of the lung microcirculatory system in the living state of the mouse, and provides a methodological basis for studies of other lung diseases in vivo.

关键词: ; 活体; 激光共聚焦显微镜

Key words: Lung; In vivo; Laser confocal scanning microscope

引用本文: 杨桢, 苏波, 钟康颖, 李岩松, 张杰. 小鼠肺部微循环活体显微成像. 华西医学, 2019, 34(1): 43-49. doi: 10.7507/1002-0179.201810029 复制

登录后 ,请手动点击刷新查看图表内容。 没有账号,
1. 张文豪, 李建军, 杨德刚, 等. 双光子显微镜在小动物活体光学成像中的研究进展. 中国康复理论与实践, 2017, 23(1): 37-41.
2. Chen X, Tang S, Zheng JS, et al. Chemical synthesis of a two-photon-activatable chemokine and photon-guided lymphocyte migration in vivo. Nat Commun, 2015, 6: 7220.
3. Li JL, Goh CC, Keeble JL, et al. Intravital multiphoton imaging of immune responses in the mouse ear skin. Nat Protoc, 2012, 7(2): 221-234.
4. Smith RA, Cokkinides V, Brooks D, et al. Cancer screening in the United States, 2010: a review of current American Cancer Society guidelines and issues in cancer screening. CA Cancer J Clin, 2010, 60(2): 99-119.
5. 包鹤龄, 方利文, 王临虹. 1990-2014 年中国 40 岁及以上人群慢性阻塞性肺疾病患病率 Meta 分析. 中华流行病学杂志, 2016, 37(1): 119-124.
6. 陆月明, 顾秀莲. 急性呼吸窘迫综合征研究进展. 中华流行病学杂志, 2005, 26(6): 463-465.
7. 成令忠, 钟翠平, 蔡文琴. 现代组织学. 上海: 上海科学技术文献出版社, 2003.
8. Lefrançais E, Ortiz-Muñoz G, Caudrillier A, et al. The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors. Nature, 2017, 544(7648): 105-109.
9. Kreisel D, Nava RG, Li W, et al. In vivo two-photon imaging reveals monocyte-dependent neutro-phil extravasation during pulmonary inflammation. Proc Natl Acad Sci USA, 2010, 107(42): 18073-18078.
10. 刘斌. 飞秒激光双光子荧光显微成像技术研究. 哈尔滨: 哈尔滨工业大学: 2012.
11. Terry RJ. A thoracic window for observation of the lung in a living animal. Science, 1939, 90(2324): 43-44.
12. Wearn JT, Ernstene AC, Bromer AW, et al. The normal behavior of the pulmonary blood vessels with observations on the intermittence of the flow of blood in the arterioles and capillaries. Am J Physiol, 1934, 109(2): 236-256.
13. Tabuchi A, Mertens M, Kuppe H, et al. Intravital microscopy of the murine pulmonary microcir-culation. J App Physiol (1985), 2008, 104(2): 338-346.
14. 夏伟强, 周源, 石明. 双光子显微成像技术的新进展. 中国医疗器械杂志, 2011, 35(3): 204-208.
15. Headley MB, Bins A, Nip A, et al. Visualization of immediate immune responses to pioneer metastatic cells in the lung. Nature, 2016, 531(7595): 513-517.
16. Vinegoni C, Aguirre AD, Lee S, et al. Imaging the beating heart in the mouse using intravital microscopy techniques. Nat Protoc, 2015, 10(11): 1802-1819.
17. 林巧雅, 黄松林, 骆清铭, 等. 肝脏免疫的活体显微光学成像研究进展. 生物化学与生物物理进展, 2017, 44(12): 1056-1065.