这项工作得到了自然科学基金项目 (81522023、81530059、91639113、81772000、81371699和 81401534) 的财政支持。

实验动物的使用由南开大学动物实验伦理委员会批准, 并按照《实验室动物护理和使用指南》进行。
1. 静电纺丝 PCL 移植物的制备
注: 本文采用静电纺丝技术制备血管移植。
<ol><li>制备 25 wt% 和 10 wt% 的 pcl 溶液, 分别在甲醇和氯仿混合物中溶解 pcl (1:5 体积比), 室温 (RT) 为12小时。</li>
	<li>将 PCL 溶液装入10毫升的玻璃注射器中。</li>
	<li>把注射器放在21克的针头上。</li>
	<li>将不锈钢芯轴 (直径2毫米, 长度25厘米) 放在收集仪器上。</li>
	<li>对于较厚的纤维移植, 使用 25 wt% 的 PCL 溶液, 从针尖到收集器17厘米的工作距离, 8 毫升/小时的流量, 以及11伏的电压作为静电纺丝参数。对于薄纤维移植, 使用 10 wt% 的 PCL 溶液, 从针尖到收集器的工作距离为20厘米, 流速为2毫升/小时, 电压为18伏, 静电纺丝参数。</li>
	<li>确保所获得的移植物被放置在真空过夜, 以消除残留溶剂。在手术前消毒所有器械, 并保持无菌技术。</li>
	<li>植入前, 通过浸泡10毫升75% 乙醇30分钟, 然后在一夜之间将其暴露在紫外线照射下, 对移植物进行消毒。</li>
	<li>纤维和孔径测量: 使用基于扫描电子显微镜 (SEM) 图像的 ImageJ 软件计算平均光纤直径。</li>
	<li>脚手架机械试验:<ol>
<li>用剃刀刀片将管状支架切成3毫米的长度段。用千分尺测量脚手架的厚度。</li>
		<li>将管状脚手架放在拉力试验机上, 负载能力为 100 N。</li>
		<li>夹紧支架与1毫米之间的钳位距离和拉纵向以10毫米/分钟的速度, 直到破裂。测量断裂时的抗拉强度和极限伸长率。从应力-应变曲线的初始线性区域 (最多5% 个应变) 计算杨氏模量。</li>
	</ol>
</li>
</ol>2. 大鼠腹主动脉植入模型
注意: 手术中使用的所有材料和仪器都是无菌的。在手术过程中, 确保操作者佩戴纱布面罩和无菌手套以避免感染。确保室温保持在 27-30 摄氏度, 以保持动物体温。遵循当地的 IACUC 指南, 关于镇痛。
<ol><li>用雄性大大鼠重 240-270 克作为血管移植的接受者。确保大鼠在手术前禁食24小时。禁食大鼠24小时的目的是充分清空肠道内的粪便, 从而拓宽操作者的视野。</li>
	<li>抓住大鼠的后颈, 把头向下, 将 springe 针插入下腹部的腹腔。用腹腔注射水合氯醛 (330 毫克/千克) 诱导大鼠麻醉。</li>
	<li>确认足够的麻醉, 确保老鼠放松肌肉和稳定呼吸。将老鼠放在手术显微镜下仰卧位。</li>
	<li>将凡士林眼科兽医软膏涂抹于眼部, 以防麻醉时干燥。使用血气生理盐水 (50 界面/毫升) 进行抗凝治疗 (100 ui/千克), 术前采用尾静脉注射。</li>
	<li>用剃刀刀片刮掉前腹壁上的皮毛, 用碘溶液和医疗酒精溶液清洁皮肤。</li>
	<li>用手术剪刀进行中线开腹切口, 确保切口长约 4-5 厘米, 然后暴露腹腔。</li>
	<li>优先用含盐水的纱布缩回和包裹肠道。</li>
	<li>仔细解剖腹主动脉。</li>
	<li>使用9-0 单丝尼龙缝线识别和结扎所有小枝。</li>
	<li>用两个血管钳夹住主动脉的隔离段 (长度可达1厘米)。主动脉可保持钳位20-30 分钟。</li>
	<li>用微型剪刀将腹主动脉与两个夹在一起, 创建吻合点。</li>
	<li>用血气盐水 (50 界面/毫升) 溶液冲洗主动脉的两端, 以去除残余血液。</li>
	<li>用微剪刀剥掉膜。</li>
	<li>吻合2毫米内径和1厘米长到大鼠腹主动脉的移植, 用9-0 单丝尼龙缝合线的八个缝合模式。</li>
	<li>首先, 根据近侧9、3、12和6点位置的顺序构造四吻合口, 然后在两条缝线之间吻合4针的切口边缘。完成近端缝合后, 用同一方法缝合远端。 注: 每针都需要确保本机侧是轻微嵌入在嫁接。</li>
	<li>取出远端钳, 让血液流入移植物, 然后取出近端钳。</li>
	<li>按缝合末端以停止使用无菌棉球或小纱布海绵止血。按压大约3分钟, 直到止血。</li>
	<li>把肠子放回腹腔。</li>
	<li>用庆大霉素 (320 U/毫升) 温热生理盐水溶液冲洗腹腔。</li>
	<li>用3-0 尼龙缝合在肌肉和皮肤层缝上腹壁, 分别。</li>
	<li>把老鼠放进干净干燥的笼子里, 在笼子下面放一个暖气垫, 保持动物体温;然后等待老鼠从麻醉中恢复。注意动物, 直到它恢复了足够的意识来维持胸骨卧床。</li>
	<li>在它恢复知觉之后, 把老鼠放进一个笼子里, 里面有食物和水。在伤口上涂上碘, 以防手术后感染。把老鼠送到其他动物的公司, 直到它完全恢复。</li>
	<li>弄死大鼠根据机构指南在预定的时间点。</li>
</ol>

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作者没有相互矛盾的经济利益。

细胞浸润对16体内血管移植的再生和重塑至关重要。有限细胞浸润通常与移植的相对较小的毛孔有关, 阻碍细胞向移植壁迁移。为了解决这一困难, 我们开发了一种改进的方法来制备静电纺丝 PCL 血管移植的大孔结构。详细地说, 孔隙大小随纤维厚度的增加而增大, 其加工参数可以很容易地控制。结果表明,在体外植入后, 宿主细胞很容易渗入到大孔移植的壁内, 细胞构?...

合成高分子的血管移植广泛应用于心血管疾病 (心血管病) 的临床治疗。不幸的是, 在小直径血管移植 (D < 6 毫米) 的情况下, 没有成功的产品, 由于低通畅触发的血液流速降低, 这往往导致血栓形成, 内膜增生, 和其他并发症1。
组织工程提供了一个替代策略, 以实现长期通畅和动态平衡的基础上的支架引导血管再生和重建。详细来说, 血管移植作为一个三维模板, 可以在血管组织再生过程中提供机械支持和结构指导, 并影响细胞功能, 包括细胞黏附、迁移、增殖和分泌细胞外基质2。到目前为止, 各种合成聚合物已被评价为应用于血管组织工程。在这些聚合物中, 聚 (己内酯) (PCL) 由于良好的细胞相容性和缓慢的降解, 从几个月到两年不等, 已被深入研究。用静电纺丝处理的大鼠主动脉模型4、5、6、PCL 血管移植表现出优良的结构完整性和通畅性, 并不断增加细胞侵袭和新生血管移植壁长达6月。然而, 不良组织重塑, 包括细胞的回归和毛细血管和钙化, 也被观察到更长的 timepoints, 长达18月。
Cellularization 血管移植是决定组织再生和重塑7的关键因素。静电纺丝作为一种多功能技术, 已广泛应用于纳米纤维结构8的血管移植的制备。不幸的是, 相对较小的孔隙结构往往导致细胞浸润不足的静电纺丝血管移植, 这限制了随后的组织再生。为了解决这一问题, 尝试了各种技术来提高孔径和整体孔隙率, 包括盐/聚合物浸出9,10, 集热器的修改, 激光辐照后处理11等。事实上, 静电纺丝的结构 (包括纤维直径、孔隙大小和孔隙度) 与加工条件12、13密切相关。在静电纺丝过程中, 通过改变聚合物溶液的浓度、流量、电压等参数, 可以很容易地控制纤维的直径. 14,15, 因此, 孔隙大小和孔隙度得到了相应的增强。
我们最近报告了一个改良的 PCL 静电纺丝移植与大孔结构 (纤维直径 5-7 µm 和毛孔 30-40 µm)。在活体移植中, 取代大鼠腹主动脉显示出高的通畅率, 以及良好的内皮化和平滑肌再生3月后手术16。更重要的是, 即使在植入一年后, 也不会有钙化和细胞回归等不良组织重塑。

<table>
	<tbody>
		<tr>
			<td>Poly(&#949;-caprolactone) (PCL) pellets (Mn=80,000)</td>
			<td>Sigma</td>
			<td>704067</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Tianjin Chemical Reagent Company</td>
			<td>1060</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol</td>
			<td>Tianjin Chemical Reagent Company</td>
			<td>1083</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Tianjin Chemical Reagent Company</td>
			<td>A1007</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Tianjin Fengchuan Company</td>
			<td>2296</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Alfa Aesar</td>
			<td>A16046</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley rats</td>
			<td>Laboratory Animal Center of the Academy of Military Medical Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td>Hebei Tiancheng Pharmaceutical company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloral hydrate</td>
			<td>Tianjin Ruijinte chemical company</td>
			<td>2223</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium Injection</td>
			<td>Tianjin Biochem Pharmaceutical company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin Sulfate Injection</td>
			<td>Jiangsu Lianshui Pharmaceutical company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-&#945;-SMA primary antibody</td>
			<td>Abcam</td>
			<td>ab7817</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-smooth MYH primary antibody</td>
			<td>Abcam</td>
			<td>ab683</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti-rat elastin antibody</td>
			<td>Abcam</td>
			<td>ab23748</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-von Willebrand factor primary antibody</td>
			<td>Abcam</td>
			<td>ab6994</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (Alexa Fluor 488)</td>
			<td>Invitrogen</td>
			<td>ab150117</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (Alexa Fluor 488)</td>
			<td>Invitrogen</td>
			<td>ab150077</td>
			<td></td>
		</tr>
		<tr>
			<td>5% normal goat serum</td>
			<td>Zhongshan Golden bridge</td>
			<td>ZLI9022</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and eosin (H&#38;E)</td>
			<td>Beijing leagene biotech</td>
			<td>DH0006</td>
			<td></td>
		</tr>
		<tr>
			<td>Masson&#39;s trichrome</td>
			<td>Beijing leagene biotech</td>
			<td>DC0032</td>
			<td></td>
		</tr>
		<tr>
			<td>Verhoeff-van Gieson (VVG)</td>
			<td>Beijing leagene biotech</td>
			<td>DC0059</td>
			<td></td>
		</tr>
		<tr>
			<td>Von Kossa</td>
			<td>Beijing leagene biotech</td>
			<td>DS0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical sutures needles with thread,3-0 silk</td>
			<td>Shanghai Jinhuan medical supplies company</td>
			<td>G3002b</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical sutures needles with thread,9-0 silk</td>
			<td>Shanghai Jinhuan medical supplies company</td>
			<td>H901</td>
			<td></td>
		</tr>
	</tbody>
</table>

implantation of electrospun vascular grafts with optimized structure in a rat model

在这里, 我们提出了一个制备大孔 PCL 血管移植的协议, 并描述了一个模型的应用腹主动脉替代模式的评估协议。静电纺丝血管移植通常具有相对较小的孔隙, 限制细胞浸润到移植物中, 阻碍新动脉的再生和重塑。本研究采用改良的工艺制备了较厚纤维 (5-6 µm) 和较大孔隙 (~ 30 µm) 的 PCL 血管移植。采用大鼠腹主动脉模型植入法对移植物的远期表现进行评价。超声分析显示, 即使植入12月后, 移植物仍未发生动脉瘤或狭窄。大孔结构改善细胞肉芽, 从而促进组织再生3月。更重要的是, 没有出现不良重塑的迹象, 例如12月后移植壁内的钙化。因此, 经改良大孔处理的静电纺丝 PCL 血管移植可作为长期植入的动脉替代物。

对 PCL 移植术后3月和12月说明, 用标准组织学技术对苏木精和伊红 (H & E)、马尾松三色、Verhoeff Gieson (VVG)、冯科萨和α SMA 的免疫荧光染色进行分析,MYH, vWF 和弹力蛋白。组织学图像采用直立显微镜, 用ﬂuorescence 显微镜拍摄免疫荧光图像。
所有数据均以平均值表示。用双尾配对学生的t检验来比较差异。p < 0.05 的值被?...

Watch this Scientific Journal Video about Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model at JoVE.com

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

优化结构静电纺丝血管移植在大鼠模型中的植入

在此, 我们提出了一种改进的静电纺丝方法, 以制造粗纤维和大孔隙的 PCL 血管移植, 并描述了一个协议, 以评估在体内的性能在大鼠腹主动脉置换模型。

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...

implantation-electrospun-vascular-grafts-with-optimized-structure-rat

Research

JoVE Journal

Biology

7.9K 次观看.  Nankai University. 在此, 我们提出了一种改进的静电纺丝方法, 以制造粗纤维和大孔隙的 PCL 血管移植, 并描述了一个协议, 以评估在体内的性能在大鼠腹主动脉置换模型。

视频: Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.

The skilled reaching scale divides the movement by a forelimb in a reach for food act into composite elements each of which are evaluated with a three-point scale. The rating scale is described for a normal rat and can be applied toward evaluating neurological motor disorders.

在鼠年达到熟练的前肢结构：一个运动评定量表

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and ...

科研

生物学

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

大鼠心脏中植入的工程组织

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

全安装胚胎肢体皮肤血管的免疫组织化学分析:一个模型系统，研究在胚胎血管分支形态发生

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

优化的基于PCR的检测支原体

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby the permeability of the endothelium to blood cells, plasma macromolecules, and water can be adapted according to the physiological need. In certain diseases, cytokines and growth factors are released that target the endothelial barrier to transiently increase vascular permeability; however, their prolonged presence may cause chronic vascular hyperpermeability and thereby tissue-damaging edema. The Miles assay is an in vivo technique that allows researchers to study vascular hyperpermeability through the proxy measurement of vascular leakage. Here, we provide a detailed protocol on how to perform this procedure in the mouse, which is the most widely used model organism to study mammalian physiology and pathology. The procedure involves the intravenous injection of Evans blue dye to label the circulating albumin followed by multiple intradermal injections of permeability-inducing agents and vehicle control solutions into opposing flanks of the mouse. Consequently, Evans blue dye gradually leaks into the dermis, where it accumulates and can be extracted for quantification as leakage induced by the permeability-inducing agent relative to the vehicle. The Miles assay can be performed in wild type or genetically modified mouse models and may be combined with drug administration to study molecular mechanisms that regulate vascular permeability and identify agents/targets capable of inducing or blocking hyperpermeability.

Here, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability.

用迈尔斯测定法评价皮肤血管 Hyperpermeability 诱导剂的含量

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

用自动光学相机进行细胞迁移的体外试验优化划痕试验

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A） 的泛化

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

近植入阶段人类胚胎中特罗福细胞的单细胞集合

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

多参数灌注对照分离原代大鼠肝细胞

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...