这项工作是由卫生部资助HL44485和HL28607国家机构的支持。

伦理声明:所有的程序进行审查和批准的机构动物护理和使用委员会。 微量移液器，限位装置和拦截1.初步加工<ol><li>使用电子牵拉调整，使得，当拉动时，在管的拉伸部分的长度约为1厘米和两半偏于对称拉在半几个清洁硼硅玻璃毛细管。确保该锥度是与图1的尺寸相一致。使用两个一半为微量，限位和阻滞剂。 <ol><li>使用斜角的气动砂轮30具有0.5微米的沐浴用水砂纸的微量。设置微量保持器的角，使其从水平约30度。 </li><li>彻底清洗，并通过使用抽吸穿过尖端和向上的轴去拉清洁丙酮干燥微量。检查微量（图1A）用一个小直立显微镜配有鳄鱼夹微量持有者和目镜标线4×和40×的目标。 </li><li>选择微量与前端开口约50μm长，有一个斜面，其长度/宽度比为3.1至3.5，并用一个尖锐的锥形点，这有助于穿透在肠系膜坚韧胶原纤维。 </li><li>存储15-20微量略微不同尺寸的在无尘框。 </li></ol></li><li>用在步骤1.1准备拉毛细管）限制部分。持拉玻璃毛细管简要地靠近一个微火以形成钝端（图1B）。存储若干在无尘框。 </li><li>用在步骤1.1准备拉毛细管）microoccluders。持拉毛细管下一个微火轻轻弯曲（使用管状适配器，例如，23G）的细尖（约4从前端毫米），使接近120度的角度，以在轴（ <s仲&gt;图1C）。制作和存储多个。 </li></ol> 2.动物准备和手术<ol><li>麻醉雄性（350-450克）或雌性Sprague-Dawley大鼠（200-300克）用戊巴比妥钠（80-100毫克/公斤，Sigma-Aldrich公司，P3761）通过皮下注射保护肠系膜不使用腹腔注射。 </li><li>在整个手术过程中和实验方案，维持麻醉根据需要补充注射（30毫克/千克）。确定由脚趾捏或角膜反射麻醉深度。的实验程序完成后，安乐死通过戊巴比妥过量或心内注射的饱和氯化钾麻醉下大鼠。 </li></ol> 3.准备解决方案和红细胞用作流标记<ol><li>制备哺乳动物林格氏溶液灌流和控制灌注溶液（通常为哺乳动物含10mg林格氏液/毫升脂肪酸游离牛血清白蛋白，BSA）中。 <ol><li>通过0.2微米注射器过滤器过滤灌流以去除可能会阻止微量移液器尖端细小颗粒;过滤成小干净的容器中。 </li></ol></li><li>通过绘制约0.2毫升的血液从尾静脉麻醉大鼠的成20G针上含有0.05 ml肝素（1000单位/毫升）的小注射器准备红细胞和转移到含约14毫升的哺乳动物林格氏溶液溶液15ml离心管。 <ol><li>离心机轻轻地收拾红细胞（约最大200×g的7分钟）。绘制上清，重悬浮红细胞在林格氏溶液中。 </li><li>离心并除去上清液后三次留在管供以后使用的底部的封装红细胞。注意，此过程减少了在最终灌流液血小板正常水平31的低于0.1％。 </li></ol></li></ol> 4.安排在动物组织盘子<ol><li>剃麻醉的老鼠的腹部从下到肚脐剑突。确保剃区域周围的右侧延伸（对于大鼠置于右侧），使得头发不会形成燃烧芯吸取灌流出组织的良好。用湿纱布或纸巾取出切下的毛发。 <ol><li>握住钝锯齿钳的皮肤，使用一个坚固的剪刀中线切口（2-3厘米长），通过皮肤。用细（IRIS）的剪刀做一个类似的切口，通过白线，解除了腹壁锯齿钳，以免损坏肠道。 </li><li>在其一侧的动物托盘上的动物，用棉签涂抹（木棍）浸泡在林格氏液和钝锯齿钳轻轻拔出从腹腔内脏。安排在组织中的肠道也使肠系膜被披在支柱观看通过显微镜注意避免接触肠系膜（POTEntially损害微血管）与任一钳或棉花。 注:动物托盘（图2A）可以从有机玻璃构成的，需要的组织以及（约3毫米深），光学透明的支柱（例如，抛光的石英约2厘米直径和5mm高），和一暖垫调整，以保持动物的体温。柱的厚度不能超过物镜的工作距离。 </li></ol></li><li>定位显微镜舞台上的动物托盘使得肠系膜是通过目镜可见。 </li><li>放置一个重力给料滴线，不断superfuse保持在35-37℃，哺乳动物的林格氏液肠系膜。用纱布垫，以保持肠道中的位置，有利于保持水分和灯芯多余的林格氏液从表面。调节流量，吸出水保持一致的灌流厚度。 </li><li>确定目标小静脉的微血管，这是收敛流，一个或两个分支远端真毛细管的下游无支链段。发现无支链的容器中（理想600〜1000微米长）具有轻快血流和游离的白细胞粘附在血管壁上。 </li><li>定位测试微血管在显微镜场的中心，并移动阻挡进入靠近选择的插管站点位置。 </li></ol> 5.填写微管和安装在支架<ol><li>就在cannulating之前，暂停红细胞的灌流液。加7微升封装红细胞的0.5 ml的灌注液在一个干净的硼硅酸盐试管。注:1-3％血球计数都可以使用，但一致的血细胞比容将​​导致从红细胞释放的任何物质一致灌洗液浓度。 </li><li>适合1毫升注射器，用23G油管接头和PE 50管（5-15厘米长）。绘制灌流/红细胞混合物倒入注射器。倒置注射器混合和驱逐所有气泡管和适配器。为了提高效率，适合管道的几个注射器在实验开始前，让他们在无尘盒或塑料袋。 </li><li>通过推进管导入微量的较大端，直到它抵靠锥形区填充微量。注射器柱塞申请一个温柔的快速推进，填补了枪头。注:涓涓溪流或滴应该是可见的尖端证据完全填充。 </li><li>拔出油管，同时轻轻推柱塞，填补了微量轴的较宽的部分。通过仔细轻弹微量轴卸下在宽轴小气泡。注意:类似的程序已经示出32。 </li><li>将微量成与侧端口，其允许连续流体连接至一个水压力计的吸移管支架。确保该保持器，其附连到用于控制微量的非常细的前进液压驱动，是在一个微小的角度（15-25度）对于水平，使得微量锥形的边缘不会撞到肠道或托盘。 </li><li>安装在一个可移动的微操作，它具有的x，y和z的驱动器，以使附近的对准到测试容器（图2）的液压驱动。 </li><li>调整使用注射器或充满水的橡胶管来改变的流体的高度，在压力计的水柱至约40 水柱肠系膜上述静水压力施加到流体中的微。 </li><li>填充吸管后尽快导管插入微血管;红细胞沉降到移液管的底部，以使大约40分钟后很少红细胞会流入容器中。 </li></ol> 6. Microcannulation和微血管压力测量<ol><li>在显微镜下定位填补微量，轻轻按下阻挡到附近施加足够的力夹住组织的微血管组织。绘制阻挡器背部，略微拉伸与微血管线的组织，以便在肠系膜胶原纤维的应力与容器对准。 <ol><li>对齐容器中的微管，降低其进入视野通过目镜观察。将吸头所选择的插管部位的上游和降低它到组织上，使得它部分地阻挡所述容器内的流动，但不会堵塞它。 </li></ol></li><li>通过驱动移液管尖端慢慢进入使用液压驱动的血管腔导管插入血管。注意不要推动通过该容器的下侧。 注意:作为微量进入内腔，灌注液迅速洗血液在血管出管腔和灌注液自由地流入到动物的循环远端测试微血管，取代某些正常血液灌注在下游容器。 </li><li>降低通过调节压力计直到血液灌注压力（所指示通过动物的红细胞）被抽回到所述灌流段;这决定了平衡的压力，其中所述红细胞轻轻摆动在血管腔和测量静水微血管压力（P c）应微血管段的远端。保持容器灌注在这个平衡压力高于一切的压力。 </li></ol> 7. Microocclusion和信息的收集<ol><li>任选的步骤:在使用微封堵以阻止容器中，将其压入在肠系膜的未使用区域中的组织远离任何容器，使尖端聚集组织的微细垫，在反复微保护实验容器之前闭塞。 </li><li>放置阻滞剂显微镜视野的下边缘附近的灌注容器的上方。 </li><li>记录有视频和音频以下。 <ol><li>口头记录块站点的位置和观察在目镜标线片的下屏幕边缘。 </li><li>与顶端阻塞置于正好微血管以上的，可使用显微的微细Ž控制轻轻降低阻滞剂直到在容器的流动闭塞。注意音频压力表压力（P C）。典型的应用闭塞3-10秒，然后松开，恢复自由的灌注。移动块站点向上朝向枪头容器中基于时间:5-10微米的步骤（&gt;初始块后5分钟，在一个位点），闭塞的数目（3-5），为防止血管壁损伤，在块站点。 </li></ol></li></ol>数据和L 的p测量8.分析（渗水） <ol><li>在视频播放期间，通过使用一个阶段微米和在图像上的已知位置的图像的确定，从一个标记红细胞的闭塞部位的初始距离（ℓ0）。在3-10秒记录图像的过程中确定它的位置在若干帧标记单元的初始速度（分升/ DT）。阻止矿容器半径（r）从阻塞（ 图3）期间拍摄的图像。 </li><li>计算ĴV / S为每个闭塞作为（分升/ DT）×（1 /ℓ0）×（R / 2）。计算部分 L P以恒定压力（j V / S）由驱动压力除以（微血管压力-有效胶体渗透压;见讨论）。 </li></ol>

The authors have nothing to disclose.

部分 L P的计算的详细情况。虽然经血管的流体运动发生同时容器自由地灌注，例如交换是太小期间自由灌注被测定，因为它通常是在容器的灌注率小于0.01％。然而，当灌注瞬时通过堵塞微血管，经血管流动停止（即，过滤）从标记红细胞在管腔作为流体的标记物红细胞和闭塞的位点之间的列中的运动测量缩短所示图3。根据实验方案的非支链容器600〜1000微米的?...

微灌在脉管嗣继承建立经由微量吸管在通常小于40微米的直径的血管受控流动的已知组合物的人工灌注液。灌注容器保持在其正​​常组织环境内，并灌注动物的血液到插管的时间。当结合的范围内的视频成象或荧光技术，原位微灌用于支持跨微血管的壁水和溶质的流动的条件下，其中为这些流动的驱动力是已知的并且在血管壁的渗透性属性可以是测量直接评估。此外，通过控制周围组织中的微血管（灌注液和灌流）的流体的组合物，微血管通透性和交换的调节可以通过使内皮细胞形成的微血管壁被暴露于各种电子商务调查xperimental条件（激动剂，修改灌注的条件下，荧光指标来衡量细胞内的成分和信令）的时间精确测量周期（秒小时）。此外，关键的细胞的分子结构调节屏障的超微结构或细胞化学评价所用的相同的微血管，其中渗透性直接测量进行调查。该方法因此形成的细胞和分子机制的研究之间的桥梁来修改内皮屏障功能中培养的内皮细胞单层和调查在完整微血管。请参阅下面的评论作进一步评估1-6。 微灌的一个限制是，它只能用于在微血管床是薄的，透明的，并具有足够的结构完整性，以使插管用玻璃微量吸管。虽然早期的研究中使用的青蛙微血管肠系膜和薄皮肤心绞痛米uscle 7,8，迄今为止最常用的制备在哺乳动物模型是大鼠肠系膜9-15。大多数研究都集中在研究过1-4小时的时期血管通透性的急性变化，但最近的调查已经扩展到测量个体船舶24-72小时的初始灌注12,16后。最近开发的CRISPR技术，这将让更多的转基因大鼠模型可用于研究血管通透性第 17条应启用该通信所描述的方法，在这些重要的新模型大鼠肠系膜小静脉的微血管被应用。 该方法要求一个倒置显微镜配备有定制的显微镜阶段足以容纳动物制备既和用于位置微型工具靠近灌注容器中并对准一个灌注微量与容器的至少三个微操作内腔。例如定制平台的xy显微镜阶段（约90×60厘米）能够从一个1厘米厚的钢板用防锈涂料来制造。该级被连接到一个工程索引表或两个燕尾滑动在水平面安装成直角并支撑在特氟隆支柱或球转移运动。一个典型的钻机（ 见图 2）有许多共同之处用于一系列的活体微循环实验，如测量单血管的血液血管平滑肌的流量和红细胞压积，局部氧输送血液灌注微血管，调节显微镜和微定位设备肌张力，并注入到整个循环荧光示踪剂的局部微血管积累。18-26 该技术的基本方面是体积流量（j v）的横跨微血管壁的规定表面面积（S）的测量。去完成这通过本文所述的改性兰迪斯技术简单的倒置显微镜是足够的。一个小的摄像机安装在图像端口和视频信号上，具有添加的时间的基础上，被显示在视频监视器上，要么在计算机上的数字形式或者作为在视频记录器的数字或模拟信号记录。一旦微血管插管微血管可见的摄像机的一部分可以通过移动台和操纵器作为一个单元而不中断插管被改变。 经血管流动的测量，也可以使用与适当的过滤器一复杂的荧光显微镜更详细的调查，如用于溶质渗透率，细胞质钙或其它细胞机制，以及共焦成像6,12,13的荧光比率监测测量钻机组合， 27。所有微灌方法的主要优点是使重复测量的能力，同船根据驱动力，如静水和肿胀压力，或引起的变化血管反应，炎症性疾病的控制变化。最常见的设计是测量水力传导率（L p）的对经由填充有控制灌注液和红细胞悬液微量第一灌注的容器相同的容器中的配对比较，以建立基线渗透性状态，然后用第二吸移管与测试试剂加入到灌注液。多插管是可能的再灌注控制吸管经过反复的恶性循环。 本协议演示了一个小静脉血管的插管和微灌大鼠肠系膜记录水通量穿过微血管壁并测量血管壁的部分 L P，公用路径的渗透性为整个完好水和溶质的一个有用的指标内皮屏障。该过程被称为改性兰迪斯techniqUE因为原来兰迪斯原理用红细胞的相对运动作为灌注被阻止被保留28后经血管的液体交换的量度的，但实验条件的范围 （例如，横跨微血管壁的静水和白蛋白肿胀压力差异）微灌后可用远比uncannulated血液灌注微血管8,29更大。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>MICROSCOPE, TABLE AND STAGE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>inverted microscope (metallurgical type) with trinocular head for video: example</td>
			<td>Olympus</td>
			<td>CK-40</td>
			<td>try to place eyepieces higher relative to stage--you have to look through eyepieces while reaching around to top of stage over intervening micromanipulators</td>
		</tr>
		<tr>
			<td>inverted microscope (metallurgical type) with trinocular head for video: example</td>
			<td>Leica</td>
			<td>DMIL</td>
			<td>try to place eyepieces higher relative to stage--you have to look through eyepieces while reaching around to top of stage over intervening micromanipulators</td>
		</tr>
		<tr>
			<td>narrow diameter, long working distance objective: example</td>
			<td>Nikon</td>
			<td>Nikon E Plan 10&#215;/0.25 LWD</td>
			<td></td>
		</tr>
		<tr>
			<td>stage platform--1/2 inch or 1 cm sheet steel</td>
			<td>welding shop</td>
			<td></td>
			<td>this should be heavy to reduce vibration</td>
		</tr>
		<tr>
			<td>Unislide x-y table: dove tail slides</td>
			<td>Velmex</td>
			<td>AXY4006W1</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VIDEO</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CCD video camera: example</td>
			<td>Pulnix</td>
			<td>TM-7CN (no longer available)</td>
			<td>no color needed</td>
		</tr>
		<tr>
			<td>video capture system with audio--generic</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>video playback system (completely still frame, single frame motion)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>small microphone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MICROMANIPULATORS, HOLDERS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator, XYZ (3)</td>
			<td>Prior/Stoelting (no longer available)</td>
			<td></td>
			<td>look for fine Z, and larger range of travel in coarse drives for ease of positioning</td>
		</tr>
		<tr>
			<td>hydraulic probe drive, one way</td>
			<td>FHC</td>
			<td>50-12-1C</td>
			<td>need to buy either manual drive or electronic drive</td>
		</tr>
		<tr>
			<td>manual drum drive&#160;</td>
			<td>FHC</td>
			<td>50-12-9-02</td>
			<td></td>
		</tr>
		<tr>
			<td>or hydraulic drive, 3 way</td>
			<td>Siskiyou Corporation</td>
			<td>MX610 (1-way) or MX630 (3-way)</td>
			<td>great for short arms, water filled and must be sent back for refill ~every 2 years</td>
		</tr>
		<tr>
			<td>connectors/rods/holders</td>
			<td>Siskiyou Corporation</td>
			<td>MXC-2.5, MXB etc.</td>
			<td></td>
		</tr>
		<tr>
			<td>pin vise</td>
			<td>Starrett</td>
			<td>162C</td>
			<td>to hold restrainer</td>
		</tr>
		<tr>
			<td>pipette holder</td>
			<td>World Prescision Instruments</td>
			<td>MPH3</td>
			<td></td>
		</tr>
		<tr>
			<td>water manometer ~120 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MICROSCOPE TRAY</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>clear Plexiglas for microscope tray for animal</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 inch polished quartz disc ~1/4 inch tall</td>
			<td>Quartz Scientific Inc.</td>
			<td>custom</td>
			<td>&#160;(or polished plexiglass, glass); make sure the height is less than working distance of objective</td>
		</tr>
		<tr>
			<td>Plexiglas glue (Weld-on 4: CAUTION CARCINOGEN)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>medical adhesive for tissue well</td>
			<td>NuSil</td>
			<td>MED-1037</td>
			<td></td>
		</tr>
		<tr>
			<td>All-purpose silicone rubber heat mat, 5&#34; L x 2&#34; W</td>
			<td>Cole Parmer</td>
			<td>EW-03125-20</td>
			<td>heater for microscope tray--needs cord and controller--240V version available</td>
		</tr>
		<tr>
			<td>Power Cord Adapter for Kapton Heaters and Kits, 6 ft, 120 VAC</td>
			<td>Cole Parmer</td>
			<td>EW-03122-75</td>
			<td></td>
		</tr>
		<tr>
			<td>STACO 3PN1010B Variable-Voltage Controller, 10 A; 120 V In, 0-140 V Out</td>
			<td>Cole Parmer</td>
			<td>EW-01575-00</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PIPET MANUFACTURE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>vertical pipette puller</td>
			<td>Sutter Instrument Company</td>
			<td>P-30 with nichrome filament</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mm OD thin wall capillary tubing</td>
			<td>Sutter Instrument Company</td>
			<td>B150-110-10</td>
			<td></td>
		</tr>
		<tr>
			<td>pipette grinder air stone and dissection microscope--see reference in text</td>
			<td></td>
			<td></td>
			<td>or purchase a package from Sutter Instruments or World Precision Instruments</td>
		</tr>
		<tr>
			<td>RX Honing Machine, System II</td>
			<td>RX Honing Machine Corporation</td>
			<td>MAC-10700 Rx System II Machine</td>
			<td>alternative for air stone, use with a dissecting microscope mounted at an angle</td>
		</tr>
		<tr>
			<td>&#160;&#160; with ceramic sharpening disc</td>
			<td>RX Honing Machine Corporation</td>
			<td></td>
			<td>use &#34;as is&#34; or attach lapping film</td>
		</tr>
		<tr>
			<td>lapping film sheets, 0.3 or 0.5 um</td>
			<td>3M</td>
			<td>part no. 051144 80827</td>
			<td>268X Imperial lapping film sheets with adhesive back--can be purchased from Amazon</td>
		</tr>
	</tbody>
</table>

microperfusion technique to investigate regulation of microvessel permeability in rat mesentery

实验测量的单独灌注微血管渗透性属性提供的调节中培养的内皮细胞单层的整个微血管床功能交换性能血管通透性分子和细胞机制调查之间的桥梁。 A到导管插入并灌注大鼠肠系膜小静脉的微血管，测量微血管壁的渗透系数方法。所需要的主设备包括一个活体显微镜用支持显微操作以定位三个不同的微型工具的大改性阶段:（1）一个斜面玻璃微量吸管到导管插入并灌注微血管; （2）的玻璃微封堵以瞬时方框灌注和使经血管水流运动的测量在测量流体静压力，以及（3）一个钝玻璃棒以稳定肠系膜组织在插管的部位。修改后的兰迪斯微闭塞techniqUE使用红细胞悬浮在人工灌注液的液体经血管运动的标志，同时也使这些流动的实验条件变化以及水压和胶体渗透压差穿过微血管的重复测量得到精心控制。第一使用控制灌注液，然后经过重新插管相同微血管与测试灌流液的水力传导率的测量，使成对的这些精心控制的条件下的微血管应答的比较。尝试的方法延伸到微血管小鼠预期修改血管通透性遗传修饰的肠系膜是因为不存在的长的直链和无支链的微血管在小鼠肠系膜严重的限制，但在大鼠相似遗传修饰的最近情况使用所述CRISPR / Cas9技术开料调查的，其中本文描述的方法可以应用的新领域。 </P&gt;

图4示出了结果从测量变化期L p的时间过程在大鼠小静脉微血管具有四个灌流液依次插管33以恒定的压力计算部分 L P的大小被用作变化微血管壁渗透性的量度，第一在用含有1％牛血清白蛋白那么当容器暴露使用含有10nM的浅滩的第二微量的抗炎剂缓激肽（BK）灌注液中的控制状态。缓激肽引起的对控制值返回在随后的10-15分钟一过性升高的部分 L P。然后...

Watch this Scientific Journal Video about Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery at JoVE.com

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

微灌技术在调查微血管渗透性的大鼠肠系膜条例

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

microperfusion-technique-to-investigate-regulation-microvessel

Research

JoVE Journal

Biology

9.7K 次观看.  University of California Davis. The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions. 

视频: Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

2 dGuo治疗胸腺器官培养的制备

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

科研

生物学

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

夹层和染色果蝇幼虫卵巢

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

大鼠肠系膜血管生成法

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

大鼠肠系膜外化:调查涉及在血管生成细胞动力学模型

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

在离体血液灌注大鼠肺备量化单微血管通透性

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

测量渗透水渗透系数（P<sub&gt; F</sub&gt;）球形细胞:隔离植物原生质体为例。

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

微灌技术在调查微血管渗透性的大鼠肠系膜条例

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