Name: microscopic observation of lymphocyte dynamics in rat peyer's patches
Uploaded: 2020-06-25T15:00:00
Description: Watch this Scientific Journal Video about Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches at JoVE.com

这项研究得到了国防医学院的资助以及日本卫生、劳动和福利省为研究棘手疾病而提供的健康和劳动科学研究补助金的支持。

实验方案经国防医学院动物研究委员会批准（第16058号）。这些动物被保存在标准实验室周（CLEA日本公司，东京，日本）。实验室动物按照国家卫生研究院的指导方针进行治疗。
1. 淋巴细胞的收集和分离
注：由于淋巴细胞必须是新鲜的，不能储存，因此每次实验都必须从大鼠那里收集淋巴细胞。此外，肠道热带淋巴细胞必须直接从它们循环的胸管中收集?...

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在这里，我们描述了收集天真的肠道热带淋巴细胞和观察它们在大鼠PPS中的迁移的协议。这些程序可以揭示淋巴细胞在PP的微血管中如何移动，并使得在正常或用药条件下对它们的动态进行视觉比较成为可能。直接观察这些动态有很多优点，以获得一些药物的免疫修饰的线索，虽然观察期仅限于几个小时。
我们在方法中提到了一些提示。其中，最微妙的程序之一是操作员必须...

佩耶的斑块（PPs）由小肠的拉米纳柱状物中的数百个淋巴状毛囊组成。PPs分为卵泡、间曲区域和位于毛囊下部的细菌中心，其中淋巴细胞受到抗原表现的刺激。没有发泄淋巴血管，抗原通过上皮细胞层从肠道流明侵入拉米纳丙酸。覆盖淋巴卵泡的上皮区域称为卵泡相关上皮，其中专门穿插M细胞吸收粘膜抗原。M细胞从发光侧摄取抗原，然后由支气管细胞捕获抗原，并呈现给通过高内皮静脉（HEV）1的内皮流入PP的天真淋巴细胞。PPs在肠道免疫中起着重要作用，与炎症的早期阶段有关。许多分子相互作用涉及淋巴细胞进入二级淋巴器官（SLOs），包括粘附分子、化疗因子2、3和磷酸-1-磷酸盐4：因此，有许多预期的治疗目标。因此，观察PP内淋巴细胞动力学使我们能够瞥见炎症的早期阶段，并检查几种有希望的药物的用处。
这里的方法侧重于PP中淋巴细胞的迁移，其中包括几个程序（可以进入胸管5， 收集淋巴细胞和注射到收集淋巴细胞后的长期观察）。由于这些程序很复杂，很难确切地看到以前的报告中每个程序是如何执行的，因此我们在这里提到了一些实现成功观察的技巧。例如，将管子插入胸管非常困难，制精的初始成功率不到50%。然而，我们改进了方法，成功率超过80%。我们在此手稿中提到了一些其他提示，这些提示对于成功观测以实现对淋巴细胞在若干条件下的跨内皮迁移进行定量评估是必要的。
在以前的报告中，很难理解随着时间的推移的三维变化，如静脉注射印度墨水染色PP6的血管结构，或显微镜是单焦7。近年来，利用凯德小鼠等一些光可转换荧光蛋白转基因动物的观察方法，澄清了体内的系统细胞运动。另一项研究澄清了CD69从PP9独立关闭淋巴细胞出口。由于其高分析能力，我们使用共分体激光扫描显微镜 （CLSM）。现在，我们可以很容易地获得高分辨率图像，并用它来分析淋巴细胞动力学。
在这份报告中，我们演示了一系列评估PP淋巴细胞迁移的方法。首先，我们展示了收集淋巴细胞的胸管罐装的精制方法。其次，我们改进了观察方法，在微观观测下尽可能保持客观器官，从而能够获得3小时的高质量图像。第三，我们量化淋巴细胞迁移的细胞运动，以评估某些药物的影响。这些修改后的协议将有助于开展粘膜免疫学评估。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">A1R+</td>
			<td style="width:213px;">Nikon</td>
			<td style="width:119px;"></td>
			<td style="width:265px;">Comfocal Laser Scanning Microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carboxyfluorescein diacetate succinimidyl ester</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">C1157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechest 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">H3570</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Wako Pure Chemical Industries</td>
			<td style="width:119px;">099-06571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 medium</td>
			<td>GIBCO</td>
			<td style="width:119px;">11875093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Texas Red&#8211;dextran</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">D1863</td>
			<td></td>
		</tr>
	</tbody>
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microscopic observation of lymphocyte dynamics in rat peyer's patches

天真的淋巴细胞在生理条件下从血液循环到淋巴组织，被公认为肠道免疫中的一个重要现象。二级淋巴细胞器官的频谱，如Peyer的斑块（PPs）或中性淋巴结，是天真淋巴细胞感知抗原的地方。天真的淋巴细胞通过血液循环，以达到高内皮静脉，进入PP的入口。据估计，一些免疫调节器会影响淋巴细胞迁移，但对微循环动力学的精确评估非常困难，建立体内淋巴细胞迁移观测方法有助于澄清精确机制。我们改进了从淋巴管中收集淋巴细胞的方法，并观察了大鼠PP中肠道淋巴细胞的详细动态。我们选择了折叠式激光扫描显微镜来观察体内的老鼠PP，并使用延时摄影进行记录。我们现在可以获得清晰的图像，有助于淋巴细胞动力学的分析。

从淋巴中收集淋巴细胞 要为大鼠准备胸管罐头，如 图1 所示，在紧张的胸管中切口，然后将大鼠维持在波尔曼的笼子中，如 图2所示。
当淋巴细胞收集良好时，我们可以获得约20 mL/6 h淋巴液，含有约107~108/mL淋巴细胞。在 TDL 中，70% 的快速 CD4，其中约 90% 的细胞表示 CD62L，这意味着 TDL 的主要组成（+6...

Watch this Scientific Journal Video about Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches at JoVE.com

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

在这里，我们描述了一个精确的方法，收集胸管淋巴细胞和观察肠道热带淋巴细胞在老鼠Peyer的补丁的迁移3小时使用延时摄影。这项技术可以澄清淋巴细胞的动态在炎症条件下是如何受到影响的。

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