Name: isolation of intrapulmonary artery and smooth muscle cells to investigate vascular responses
Uploaded: 2022-06-08T13:30:00
Description: Watch this Scientific Journal Video about Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses at JoVE.com

作者要感谢泰国国家研究委员会，化学创新卓越中心（PERCH-CIC）和国际研究网络（IRN61W0005）提供财政支持，以及纳瑞宣大学生理学系医学科学学院的研究设施支持。

本研究中进行的实验已获得Naresuan大学动物护理和使用委员会（NUACUC）伦理委员会的批准，协议号为NU-AE620921，用于科学目的的动物护理和使用。
1.生理溶液的组成
<ol><li>通过将化学物质溶解在蒸馏水中来配制克雷布斯溶液，以达到以下最终浓度：122 mM NaCl、10 mM HEPES、5 mM KCl、0.5 mM KH 2 PO 4、0.5 mM NaHPO4、1 mM MgCl 2、1.8 mM CaCl 2 和 11<s...

<ol>
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U., Ingkaninan, K., Waranuch, N., Chootip, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasodilatory+effects+and+mechanisms+of+action+of+Bacopa+monnieri+active+compounds+on+rat+mesenteric+arteries.">Vasodilatory effects and mechanisms of action of Bacopa monnieri active compounds on rat mesenteric arteries.</a> Molecules. 24 (12), 2243 (2019).</li><li>Chootip, K., Kennedy, C., Gurney, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+P2+receptors+mediating+contraction+of+the+rat+isolated+pulmonary+vasculature.">Characterization of P2 receptors mediating contraction of the rat isolated pulmonary vasculature.</a> British Journal of Pharmacology. 131, 167 (2000).</li><li>Paracha, T. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidation+of+vasodilation+response+and+structure+activity+relationships+of+N2,+N4-disubstituted+quinazoline+2,+4-diamines+in+a+rat+pulmonary+artery+model.">Elucidation of vasodilation response and structure activity relationships of N2, N4-disubstituted quinazoline 2, 4-diamines in a rat pulmonary artery model.</a> Molecules. 24 (2), 281 (2019).</li><li>Chootip, K., Gurney, A. 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在这篇手稿中，我们描述了分离大鼠IPA和VSMC的技术。已经采用了几种实验方案来研究体 外IPA的血管反应，可用于表征植物提取物诱导的IPA血管松弛的药理作用和机制基础。
关于植物提取物的内皮依赖性血管扩张作用，采用了各种阻断剂，如L-NAME（eNOS），吲哚美辛（COX）和apamin+charybdotoxin（EDHF）。代表性数据显示阳性结果（即，在存在eNOS或EDHF抑制剂的情况下血管扩张...

肺脉管系统是一种低压血管系统，其主要功能是将脱氧血液输送到肺部的气体交换区域。肺中的肺动脉排列在平行于支气管树的分支中，最终形成一个广泛的毛细血管网络，该网络在几个肺泡上连续，最后汇聚成小静脉和静脉。肺动脉的血管张力受多种因素控制，涉及内皮和血管平滑肌细胞（VSMC）之间的相互作用1。
在这项研究中，我们专注于肺内动脉（IPA）的内皮依赖性和非依赖性血管松弛。关于内皮依赖性血管松弛，内皮细胞表面发生的各种机制可以增加细胞内Ca2+浓度（例如乙酰胆碱[ACh]与毒蕈碱受体[M3]结合），导致一氧化氮（NO），前列环素（PGl 2）和内皮衍生的超极化因子（EDHF）的形成（图1).NO是主要的内皮衍生松弛因子，由L-精氨酸通过内皮一氧化氮合酶（eNOS）2合成，然后从内皮细胞解离为VSMC（图1）并刺激可溶性鸟苷酸环化酶（sGC）酶;这种酶将鸟苷三磷酸（GTP）转变为环鸟苷单磷酸（cGMP），其激活蛋白激酶G（PKG）并降低胞质Ca 2+水平，从而导致血管松弛（图1）。PGl 2 由内皮细胞通过环加氧酶 （COX） 途径3，4 合成。它与VSMC上的前列环素受体（IP）结合并刺激腺苷环化酶（AC）酶，然后将三磷酸腺苷（ATP）转化为环磷酸腺苷（cAMP）（图1）3，4。cAMP激活蛋白激酶A（PKA），降低胞质Ca2+水平并引起血管松弛5（图1）。EDHF通路还通过各种内皮介质和电事件参与内皮依赖性血管松弛。EDHF途径的激活导致VSMC的超极化，从而关闭电压操作的Ca 2+通道（VOCC），降低细胞内Ca 2+水平，并诱导血管松弛6。内皮非依赖性血管松弛通过多种机制直接发生在VSMC上，例如细胞内Ca 2+水平的降低，肌球蛋白轻链激酶（MLCK）的抑制，肌球蛋白轻链磷酸酶（MLCP）的活化以及Ca2+对VSMC收缩机制的敏感性降低。在本研究中，我们重点关注各种K+通道的打开，VOCC的阻断以及抑制肌质网7释放的Ca 2+引起的血管松弛，导致细胞内Ca 2+水平降低，从而分别降低VSMC肌球蛋白轻链磷酸化和肌球蛋白-肌动蛋白结合或交叉桥形成， 最终导致血管松弛。
在孤立的IPA中评估血管收缩和血管松弛测量的技术对于啮齿动物来说已经很成熟，但数据因实验方案而异。本研究描述了用于评估 体外大鼠IPA制剂血管反应性的方法，该方法是在没有调节 体内血管反应的外部因素（如神经信号、激素、细胞因子、血压等）的情况下进行的。
我们采用几种实验方案，以植物提取物为例来研究IPA的血管反应性。使用各种阻断剂（图1）来确定植物提取物诱导的内皮依赖性和非依赖性血管松弛的机制。然而，相同的方案可以用于评估IPA对用于治疗各种肺部病变的任何药物，提取物或植物化学物质的血管反应。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,4-dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>D0632 
			CAS NO. 348-12-3</td>
			<td></td>
		</tr>
		<tr>
			<td>4-aminopyridine (4-AP)</td>
			<td>Aldrich Chemical</td>
			<td>A78403 
			CAS NO. 504-24-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetylcholine</td>
			<td>Sigma-Aldrich</td>
			<td>A6625 
			CAS NO. 60-31-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Apamin</td>
			<td>Sigma-Aldrich</td>
			<td>A9459 
			CAS NO. 24345-16-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2153 
			CAS NO. 9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium choride</td>
			<td>Ajax Finechem</td>
			<td>AJA960 
			CAS NO. 1707055184</td>
			<td></td>
		</tr>
		<tr>
			<td>Charybdotoxin</td>
			<td>Sigma-Aldrich</td>
			<td>C7802 
			CAS NO. 95751-30-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type 1A</td>
			<td>Sigma-Aldrich</td>
			<td>C9891 
			CAS NO. 9001-12-1</td>
			<td>From Clostridium histolyticum</td>
		</tr>
		<tr>
			<td>D(+)-Glucose monohydrate</td>
			<td>Millipore Corporation</td>
			<td>K50876942 924 
			CAS NO. 14431-43-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D4540 
			CAS NO. 67-68-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis (2-aminoethylether)-N,N,N&#8217;,N&#8217;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E3889 
			CAS NO. 67-42-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E9884 
			CAS NO. 60-00-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps 11 cm.</td>
			<td>Rustless Dumoxel</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps 14 cm.</td>
			<td>Rustless Dumoxel</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Glibenclamide</td>
			<td>Sigma-Aldrich</td>
			<td>G6039 
			CAS NO. 16673-34-0</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism program</td>
			<td>Software version 5.0 (San Diego, CA, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375 
			CAS NO. 7365-45-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Iberiotoxin</td>
			<td>Sigma-Aldrich</td>
			<td>I5904 
			CAS NO. 1002546960</td>
			<td>recombinant from Mesobuthus tamulus</td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma-Aldrich</td>
			<td>I7378 
			CAS NO. 53-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Labchart Program</td>
			<td>Software version 7.0 (A.D. Instrument, Castle Hill, Australia).</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Ajax Finechem</td>
			<td>296 
			CAS NO. 1506254995</td>
			<td></td>
		</tr>
		<tr>
			<td>Male Wistar rats</td>
			<td>Nomura Siam International Co. Ltd., Bangkok, Thailand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NG-nitro-L-arginine methyl ester (L-NAME)</td>
			<td>Sigma-Aldrich</td>
			<td>N5751 
			CAS NO. 51298-62-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicardipine</td>
			<td>Sigma-Aldrich</td>
			<td>N7510 
			CAS NO. 54527-84-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Organ bath 15 mL.</td>
			<td>-</td>
			<td>-</td>
			<td>Specific order by the researchers</td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Sigma-Aldrich</td>
			<td>P4762 
			CAS NO. 9001-73-4</td>
			<td>FromPapaya Latex</td>
		</tr>
		<tr>
			<td>Phenal red</td>
			<td>Sigma-Aldrich</td>
			<td>P5530 
			CAS NO. 34487-61-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylephrine</td>
			<td>Sigma-Aldrich</td>
			<td>P6126 
			CAS NO. 61-76-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Kemaus</td>
			<td>KA383 
			CAS NO. 7447-40-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogenphosphate</td>
			<td>Aldrich Chemical</td>
			<td>EC231-913-4 
			CAS NO. 7778-77-0</td>
			<td></td>
		</tr>
		<tr>
			<td>S+A2:E36odium chloride</td>
			<td>Kemaus</td>
			<td>KA465 
			CAS NO. 7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors 11 cm.</td>
			<td>Spall Stainless</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors 14 cm.</td>
			<td>Spall Stainless</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Ajax Finechem</td>
			<td>475 
			CAS NO. 912466</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogenphosphate</td>
			<td>Aldrich Chemical</td>
			<td>33,198-8 
			CAS NO. 7558-80-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Ajax Finechem</td>
			<td>482 
			CAS NO. 1506196602</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thiopental</td>
			<td>Anesthal</td>
			<td>JPN3010002 
			CAS NO. 1C 314/47</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625 
			CAS NO. 107-35-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Waterbath WBU 45</td>
			<td>Memmert</td>
			<td>2766 
			CAS NO. -</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of intrapulmonary artery and smooth muscle cells to investigate vascular responses

从大鼠肺分离的肺内动脉（IPA）和血管平滑肌细胞（VSMC）可用于研究血管收缩和血管松弛的潜在机制。分离IPA和VSMC后，可以在没有神经信号，激素，细胞因子等外在因素的情况下评估生理和病理条件下血管反应的特征。因此，IPA和VSMC是研究血管生理学/病理生理学以及各种实验研究的优秀模型，例如药物调节，膜片钳电生理分析，钙成像等。在这里，我们使用了一种分离IPA的技术来研究器官浴设置中的血管反应。通过腔内导线将IPA段安装在器官浴室上，并受到各种药物的刺激。使用等长力传感器和生理数据分析软件程序记录IPA血管张力的变化（即血管收缩和血管松弛）。我们实施了几种实验方案，可以调整用于研究血管松弛/血管收缩的机制，以研究植物化学或合成药物的药理活性。这些协议还可用于评估药物在调节各种疾病中的作用，包括肺动脉高压。IPA模型使我们能够研究浓度-反应曲线，这对于评估药物的药效学参数至关重要。

本研究中的方案已被开发用于确定测量在分离的IPA制剂的血管反应中观察到的生理现象的最佳实验条件。进行试点实验以描述有助于理解植物提取物血管松弛作用的血管效应和机制基础的潜在结果，如下所示。
植物提取物的血管松弛作用 如图6A，B所示，在内皮完整IPA（E +）中，植物提取物引起血管松弛的浓度依赖性反应（EC<...

Watch this Scientific Journal Video about Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses at JoVE.com

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

动脉肺循环的血管反应可以使用肺内动脉 （IPA） 和血管平滑肌细胞 （VSMC） 进行探索。本研究详细描述了IPA的分离以及用于研究对生理刺激的反应的血管松弛的方案。

分离肺内动脉和平滑肌细胞以研究血管反应

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of ...

本研究描述了分离大鼠肺内动脉和血管平滑肌细胞。采用了几种实验方案，例如使用器官浴技术来研究肺内动脉的血管反应。肺内动脉和血管平滑肌细胞是研究血管生理学和病理生理学的极好模型。该技术的改编可以评估任何药物，提取物或植物化学物质的血管反应。将孤立的肺内动脉环安装在器官浴中，可以使用孤立的肺内动脉来评估药物在调节各种疾病（包括肺动脉高压）中的作用。实验方案在技术上是可行的，但关键步骤很复杂，对于成功至关重要，因此进行视觉演示使其更容易理解和遵循。演示该程序的是Naresuan大学医学院生理学系心血管研究单元实验室的生理学硕士生Kittiwoot To-on。首先用11厘米的剪刀切开肺的单个叶，并将其放在9厘米的培养皿上，内侧或肺根朝上。从上到下观察并确定静脉、支气管和动脉的排列情况。用剪刀纵向切开支气管。然后用11厘米长的镊子抓住支气管的尖端。轻轻解剖并将支气管和静脉从肺部取出。用镊子抓住肺内动脉的尖端，用剪刀小心地将其从肺组织中解剖出来。将孤立的肺内动脉保持在冷克雷布斯溶液中，直到器官浴组件建立起来。隔离肺内动脉后，用11厘米的剪刀纵向切开肺内动脉的主分支，切成2毫米的条状。将肺内动脉试纸浸入DM中，然后将试纸在含有每毫升木瓜蛋白酶1毫克，0.04%BSA和0.4毫摩尔DTT的DM中在4摄氏度下孵育一小时。然后在 37 摄氏度下孵育 15 分钟。在DM中每毫升加入一毫克的IA型胶原酶，并再次在37摄氏度下孵育五分钟。将组织转移到新鲜的DM中，并使用玻璃巴斯德移液管轻轻研磨分散。继续研磨，直到在显微镜下的沐浴液中可见孤立的血管平滑肌细胞。如前所述隔离肺内动脉，并将肺内动脉的主要分支切成约2毫米长的环。通过将肺内动脉环穿在两根直径为 40 微米的不锈钢丝上，将它们固定在器官浴室中。将装有肺内动脉环的不锈钢线连接到连接到数据采集装置的等长力传感器和安装了用于数据记录和分析的合适生理软件的计算机系统。然后轻轻地将肺内动脉环的张力提高到一克。让血管段在 1 克的静息张力下平衡约 45 分钟。在平衡期间，确保克雷布斯溶液每15分钟定期更换一次。平衡后，通过测量血管收缩到高细胞外钾溶液来测试血管的活力。通过计算用PE预收缩的环中对乙酰胆碱的松弛反应来评估内皮的存在与否。通过用小线轻轻摩擦血管内部以诱导剥蚀来机械地去除内皮。然后在测试实验开始前平衡动脉环30分钟。通过用PE预先收缩肺内动脉环来研究植物提取物的松弛作用。然后小心地将每毫升0.1至1000微克的植物提取物累积添加到内皮完整环和内皮剥离环中，以诱导血管松弛并获得浓度依赖性反应曲线。确保用作溶剂的有效DMSO也经过类似的评估，以用作阴性对照。评估文本手稿中提到的植物提取物的血管松弛作用机制。然后在收缩到PE稳定后，加入累积浓度的植物提取物。将植物提取物的效果呈现为存在抑制剂时肺内动脉环的松弛百分比与无抑制剂的肺内动脉环的反应相比，并构建浓度反应曲线。在内皮完整肺内动脉中，植物提取物引起血管松弛的浓度依赖性反应。根除内皮细胞可显著降低植物提取物诱导的血管松弛，EC50增加2.2倍，eNOS和EDHF抑制明显降低植物提取物的血管松弛反应。这会将浓度响应曲线向右移动，并在不改变E最大值的情况下增加EC50。相反，吲哚美辛对植物提取物的血管松弛剂反应没有影响。在内皮剥落的肺内动脉环中，钙激活的钾通道阻滞剂降低了对植物提取物的血管松弛反应。而电压门控或ATP敏感钾通道的阻断剂没有显示偏差。与植物提取物一起预孵育抑制了氯化钙诱导的收缩。内皮剥去的肺内动脉环与无钙克雷布斯溶液和 PE 预孵育，呈现短暂收缩。与车辆相比，植物提取物显着减少了PE引起的收缩。在手术过程中，必须尽早清楚地识别静脉、支气管和动脉。切割或损伤动脉会减少其长度并影响血管反应。该技术可用于研究可能作为肺血管疾病（如肺动脉高压）治疗原型的化合物。

Research

JoVE Journal

Medicine

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

肠系膜动脉收缩和舒张的研究，使用自动线Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

科研

医学

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

培养一个精密的显微外科技术在大鼠颈总动脉，颈外静脉移植：介入

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

右心室结构和功能的肺动脉缩窄经胸超声小鼠模型的评估

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

膀胱平滑肌收缩带作为一种方法来评估下尿路药理学

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

血管球囊损伤和腔内管理在大鼠颈动脉

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

左冠状动脉前降支的小鼠结扎：对心肌梗死后重塑和心衰模型

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

冠脉流量和冠状动脉血流储备用小鼠压力超负荷基于模型的超声评估

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

三氯化铁诱发血栓形成小鼠模型上的颈动脉和肠系膜血管

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

主动脉钙化和炎症血管平滑肌细胞和成像的钙化

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...