Name: intratracheal instillation of stem cells in term neonatal rats
Uploaded: 2020-05-04T14:30:00
Description: Watch this Scientific Journal Video about Intratracheal Instillation of Stem Cells in Term Neonatal Rats at JoVE.com

这项研究得到了台湾台北美利根生物科技有限公司（A-109-008）的赠款的部分支持。

这一程序得到了台北医科大学动物护理与使用委员会的批准。
注：人类MSC与绿色荧光蛋白（GFP）和萤火虫荧光酶基因（Fluc）稳定地转染，这些基因是从一家商业公司（材料表）获得的。
1. 具有萤火虫荧光酶和绿色荧光蛋白的人类MSC特征
<ol><li>在37°C的饱和湿度和5%的CO2中，保持与GP和F1的人类MSC在完整介质中（最低必需的中等鹰-阿尔法修饰[...

<ol>
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患有呼吸窘迫的新生儿通常需要腹内表面活性剂和/或皮质类固醇治疗19。人类第一阶段的临床试验已经证明了早产儿8期中腹间MSC的安全性。这些研究表明，对患有呼吸窘迫的新生儿来说，在血管内服用药物是一个重要的选择。如果模型特征与人类直接相关，动物模型研究最有帮助。定期新生大鼠是早产肺损伤和发育研究的有用模型。然而，新...

治疗患有呼吸窘迫的新生儿时，通常需要补充氧气。然而，婴儿的超氧治疗有长期不利影响。长时间接触高浓度的氧气会导致炎症和急性肺损伤，这类似于人类支气管肺发育不良（BPD）2。BPD 是高氧治疗的主要并发症，尽管早产儿有早期表面活性剂治疗、最佳的通气程序以及使用非侵入性正压通气的增加，但可能会发生这种情况。虽然许多治疗策略已经报告为BPD3，没有已知的治疗可以减少这种并发症。
皮质类固醇的使用是预防BPD的一种潜在治疗方法，因为肺炎在其发病机制中起着至关重要的作用。然而，由于长期不良反应,4，5，通常不建议早产儿进行全身皮质类固醇治疗。4
脑膜状细胞（MSCs）具有多能性特征，可以分化成各种细胞类型，包括骨骼、软骨、脂肪组织、肌肉和肌腱6。MSC具有免疫调节、抗炎和再生作用7，动物研究表明MSCs及其分泌成分在啮齿动物8、9,中高氧引起的肺部损伤中具有治疗作用。内切和静脉MSC移植已被证明可以预防新生儿高氧肺损伤。因此，干细胞的腹内给药和表面活性剂和皮质类固醇联合治疗可能是治疗新生儿呼吸系统疾病的潜在治疗策略。临床前研究在新生大鼠10、11、12,11中使用了干细胞和腺相关病毒的腹内给给。然而，该技术的分步演示和移植干细胞的体内跟踪是不可用的。新生大鼠适合研究腹内给药对患有呼吸窘迫的早产儿的影响，因为大鼠出生时肺的囊期相当于妊娠13的26-28周的人肺的囊期。一种有效的方法，为大鼠气管的药而，是成功进行肺分配的关键。这里介绍的技术允许研究细胞和/或药物的腹内给药，以大鼠为人类模型治疗新生儿肺病。

<table>
	<tbody>
		<tr>
			<td>6-0 silk</td>
			<td>Ethicon</td>
			<td>1916G</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol Prep Pad</td>
			<td>CSD</td>
			<td>3032</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Stemflow hMSC Analysis Kit</td>
			<td>BD Biosciences</td>
			<td>562245</td>
			<td>CD markers</td>
		</tr>
		<tr>
			<td>CMV-Luciferase-EF1&#945;-copGFP BLIV 2.0 Lentivector for In Vivo Imaging</td>
			<td>SBI</td>
			<td>BLIV511PA-1</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoStor10</td>
			<td>BioLife Solutions</td>
			<td>640222</td>
			<td></td>
		</tr>
		<tr>
			<td>Human MSCs</td>
			<td>Meridigen Biotech Co., Ltd. Taipei, Taiwan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Infrared light</td>
			<td>JING SHANG</td>
			<td>JS300T</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Halocarbon</td>
			<td>26675-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS-200 small animal imaging system</td>
			<td>Caliper LifeSciences, Hopkinton, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferin potassium salt</td>
			<td>Promega, Madison, WI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-scissors, straight</td>
			<td>Vannas</td>
			<td>H4240</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td>TAIWAN BIOTECH CO., LTD.</td>
			<td>113531</td>
			<td>Isotonic Sodium Chloride Solution</td>
		</tr>
		<tr>
			<td>Small Hub RN Needle, 30 gauge</td>
			<td>Hamilton Company, Reno, NV</td>
			<td>7799-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (100 &#181;l)</td>
			<td>Hamilton Company, Reno, NV</td>
			<td>81065</td>
			<td></td>
		</tr>
		<tr>
			<td>Xenogen Living Image 2.5 software</td>
			<td>Caliper LifeSciences, Hopkinton, MA</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

intratracheal instillation of stem cells in term neonatal rats

长时间接触高浓度的氧气会导致炎症和急性肺损伤，这类似于人类支气管肺发育不良（BPD）。在早产儿中，尽管早期使用表面活性剂疗法、最佳通气策略和非侵入性正压通气，但BPD是一个主要并发症。由于肺炎在BPD的发病机制中起着至关重要的作用，皮质类固醇的使用是预防它的潜在治疗方法之一。然而，由于长期不良反应，通常不建议早产儿进行全身皮质类固醇治疗。临床前研究和人类第一阶段临床试验表明，在高氧引起的肺损伤和早产儿中使用间质期频闪细胞（MSCs）是安全和可行的。内切和静脉MSC移植已被证明可以预防新生儿高氧肺损伤。因此，干细胞的腹内给药和表面活性剂和糖皮质激素联合治疗已成为治疗新生儿呼吸系统疾病的新策略。大鼠肺出生时的发育阶段相当于妊娠26~28周时人类肺的发育阶段。因此，新生大鼠适合研究腹内给患呼吸窘迫的早产儿，以评估其疗效。这种中切内注射技术是将干细胞和药物移植到肺部的临床可行选择。

新生儿大鼠中干细胞的脑内注入的肺分布由萤火虫荧光酶（Fluc）标记的干细胞确定。MSC 被标记为 Fluc，并通过延日病毒转导标记绿色荧光蛋白。 图1A 显示人类MSC中的GPC表达水平很高，93.7%的种群显示流量细胞学检测到的GP阳性表达。MSC的特点是分析CD标记（即CD 44、CD73、CD90和CD105）的表达，以及分线分化成骨细胞、软细胞和脂肪细胞的能力（图1B?...

Watch this Scientific Journal Video about Intratracheal Instillation of Stem Cells in Term Neonatal Rats at JoVE.com

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

描述是通过期期新生儿大鼠的四边形注射进行间质质质细胞（MSCs）的腹内移植的协议。这项技术是将干细胞和药物移植到新生儿大鼠肺以评估其疗效的临床可行选择。

期新生儿大鼠干细胞的内切内培养

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia ...

内切注射是一种临床生物技术，用于将干细胞和药物移植到新生儿肺部，以评估Chilman的疗效。这种技术在新生儿大鼠的各种初级应用中可能非常适用，是一种相对容易且具有成本效益的肺病治疗措施。产后第五天，在60度角的插管站上，对斯普拉格-道利大鼠幼崽进行麻醉。并在每只小狗的所有四肢上贴上实验室标签胶带，以确保它们就位。在每个鼻子下面涂抹胶带来固定头部。并精确定位穿刺气管切除术的第一个颈部。使用 75% 酒精准备垫对切口区域进行消毒。并在气管上方进行垂直的3厘米中线切口，以避免损坏管状动脉。使用弯曲的锥形钳子，没有挂钩，以分离出脂肪和肌肉层，以定位气管。当可以观察到气管时，使用钳子抓住气管，并使用装有30量针的100微升注射器，在吸入阶段将第五发蝇荧光素酶GP的十倍注射，在30微升盐水中标记的间质热细胞进入气管。当所有细胞都注射后，用160根丝线将结尽可能紧密地绑住切口。尽可能短切线。然后让幼崽从麻醉中恢复过来，在温暖的地方进行监测，直到它温暖、粉红色，并且能够自发运动，然后再将动物返回到笼子里。为了跟踪移植的人类中质体细胞在大鼠从手术中恢复15分钟后，在PBS中注射每公斤125毫克路西法钾盐的再麻醉幼崽。注射后 10 分钟，使用小型动物成像系统以 5-15 秒的间隔获取顺序图像，并采用中等装箱、1 f-stop 和 26 厘米的视野。然后使用成像软件根据感兴趣的自动区域量化肺部的发光活动。在标有人类中层干细胞的荧光素酶萤火虫中观察到高水平的GP表达。中质干细胞可以进一步的特点是其细胞表面标记表达和它们区分成骨细胞、软骨细胞和脂肪细胞的能力。VIVO中移植的人类中质细胞的发光成像显示，用正常盐水治疗的大鼠的肺区域缺乏发光信号。在用中质干细胞治疗的大鼠中，在这些动物的气管和中肺区域观察到发光。事实上，在这个具有代表性的分析中，对发光强度的量化表明，与用正常盐水治疗的老鼠相比，用间质干细胞治疗的大鼠的发光活性增加了约13倍。尝试此程序时，在不限制周围动脉的情况下解剖和隔离气管非常重要。该技术允许研究细胞内注射和新生儿帕尔默病药物的影响，使用射线与人类患者的模式。

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Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

在新生儿猪淋巴的肠淋巴躯干和收集的长期导尿

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

A Neonatal Mouse Spinal Cord Compression Injury Model

新生儿小鼠脊髓压迫损伤模型

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

在P10大鼠新生儿中风的短暂大脑中动脉栓塞模型

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

毛喉素诱导在肠组织体溶胀：一个<em&gt;体外</em&gt;检测方法的评估囊性纤维化患者对药物的反应

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

大鼠双侧髌腱损伤模型中干细胞治疗的评价

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

聚丙烯酸/纳米水的气管内灌注积液模型

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...