这项工作是支持OTKA（匈牙利科学研究基金）D45933，T049621，TET（匈牙利科学与技术基金会）A4/04，Arg-17/2006和单仲偕，Bolyai，Öveges奖学金和TÁMOP 4.2.2-08 / 1 / KMR - 2008 - 0004和4.2.1 / B 09/1/KMR-2010-0001。 OTKA 83803。我们想感谢威廉Gesztes提供语音。

1。准备H9C2细胞cardiomyoblast H9C2大鼠cardiomyoblasts取自ATCC（德国Wesel的，）和扩展的DMEM含10％胎牛血清，4毫米L -谷氨酰胺，100 U / ml青霉素和100μg/ mL链霉素的高糖（4.5克/升）。 H9C2成肌细胞线是从胚胎大鼠心脏产生的，它是用来作为一个在 8,9两个骨骼肌和心肌的体外模型。 准备H9C2细胞的12孔板: <ol><li>删除的Petri菜H9C2细胞培养液中。 </li><li>两次2-3毫升0.05％胰蛋白酶/ EDTA清洗用5 ml PBS和消化细胞。 </li><li>将培养皿在37℃培养箱中培养5分钟。 </li><li>用10 ml DMEM培养液抑制胰蛋白酶。 </li><li>在8分钟1200转离心细胞。 </li><li>取出后在1毫升的DMEM培养液和使用台盼蓝染色血球计数细胞上清，重悬细胞。</li><li>种子30000 H9C2细胞在DMEM培养液2毫升，每孔在12孔板。 </li><li> 24小时，放入37℃二氧化碳培养箱板。 </li></ol> 2。模拟缺血/再灌注缺血/再灌注是在体外模拟执行H9C2细胞培养氧糖剥夺（OGD）。此过程是PeCon细胞的孵化体系，使观察细胞在OGD（德国Erbach，）蔡司共聚焦显微镜的阶段。 <ol><li>卸下H9C2细胞的12孔板的愿望语言。 </li><li>用PBS洗涤细胞两次。 </li><li>加入3毫升葡萄糖培养基，每孔。 </li><li>将进入细胞孵育系统板。 </li><li>启动氮气吹扫孵化体系的氧气。 </li><li>等待，直到氧气水平达到0.5％，然后开始计时。 0.5％的氧气是指局部紧张约3毫米汞柱。除细胞为1模拟缺血50分钟。 </li><li>模拟缺血结束，从细胞中移除的葡萄糖培养基。 </li><li>加入2毫升正常培养基井。 </li><li>放置30分钟，在37℃二氧化碳培养箱12孔板，这是"再灌注期" 。 </li></ol> 3。准备抢救间质干细胞人类起源的骨髓是采取到T75烧瓶和贝科的修改鹰的中等（DMEM）文化介质含10％胎牛血清（FCS），100单位/毫升青霉素和100微克/毫升链霉素，4 mM的大号-谷氨酰胺和稀释1 g / L葡萄糖。在37 ° C孵育烧瓶，在充分湿润的5％的CO 2，连续3天的气氛。用PBS洗涤后的潜伏期坚持瓶和骨髓其余的组件表面的骨髓基质干细胞被淘汰。用骨髓基质干细胞在实验1和5之间的通道。该IDENtity细胞被证实存在系特异性的细胞表面标志物，用流式细胞仪。造血行数的特异性表面标志（CD34，CD45）和间质的表面标志物（CD73，CD90，C​​D105和CD166）进行了调查。 在模拟缺血，准备抢救间质干细胞移植。 <ol><li> Vybrant在1:200比例的DMEM培养液稀释荧光染料。 </li><li>吸救援细胞的培养基，添加300μL的DMEM与Vybrant。 </li><li>将培养皿在37℃ 二氧化碳培养箱30分钟。 </li><li>删除的Petri盘间质干细胞的培养基中。 </li><li>两次2-3毫升0.05％胰蛋白酶/ EDTA清洗用5 ml PBS和消化细胞。 </li><li>将培养皿在37℃培养箱中培养5分钟。 </li><li>用10 ml DMEM培养液抑制胰蛋白酶。 </li><li>在8分钟1200转离心细胞。 </li><li>取出后的SUPErnatant 1毫升的DMEM培养液和使用台盼蓝染色血球计数细胞，悬浮细胞。 </li><li> 30分钟，再灌注结束后增加20000间质干细胞，每孔缺血心脏肌细胞H9C2。 </li><li>共培养的细胞放入37℃ 二氧化碳培养箱24小时。 </li></ol> 4。共聚焦显微镜分析模拟缺血/再灌注损伤后，细胞在24小时正常培养基培养。 <ol><li> 24小时后，用PBS洗涤细胞两次。 </li><li> 500μL活/死30分钟（5纳米钙黄绿素和400 nm乙锭- 2 -二聚体在PBS）的染料溶液（5X10 4细胞/毫升）染色细胞。 </li></ol>评价选定的生活和死亡的细胞形态和荧光共聚焦图像可以执行与ImageJ软件（国立卫生研究院，美国）。在CO型文化，MSCS可区别于H9C2细胞，由于其Vybrant细胞标记。评估的死细胞比例可在4个独立的领域的观点（客观10X）为每一种文化在盲目的时尚 10 。 42毫米的盖玻片培养的H9C2 cardiomyoblasts也可以与时间的推移视频显微镜进行调查期间和之后，缺血/再灌注损伤，使用共聚焦显微镜观察细胞孵化体系。随着时间的推移，可以研究细胞与细胞之间的相互作用，如细胞融合，形成间碳纳米管。 5。用流式细胞仪分析模拟缺血/再灌注损伤后，细胞在24小时正常培养基培养。 <ol><li> 24小时后，收获的培养基。这是收集培养液中也非常重要，因为一些死细胞可以从培养皿表面分离，在24小时incubaTION。 </li><li>两次洗净，用PBS和消化细胞，用0.05％胰酶EDTA。 </li><li>抑制胰蛋白酶，DMEM培养液。 </li><li>降速1200 RPM（约150 - 200G）的细胞和收获的培养基为8分钟。 </li><li>弃上清。 </li><li>暂停500μL的活/死30分钟（5纳米钙黄绿素和400 nm乙锭- 2 -二聚体在PBS）的染料溶液（5X10 4细胞/毫升）细胞。 </li><li>转移细胞，流式细胞仪检测管。 </li><li>启动CellQuest Pro软件。 </li><li>要成立流式细胞仪进行测量，准备控制活细胞和死细胞样本。 <ol class="lalpha"><li>在第1点所述板H9C2细胞在12孔板密度3x10 4。 </li><li>准备生活与单trypsinisation细胞控制，如第1点所述。 </li><li> 1小时10微米H 2 O 2处理准备死细胞控制。 </li><li>后trypsinisation，resuspenð活的和死细胞控制在500μL活/死染料溶液（5纳米钙黄绿素- AM和400 nm乙锭-二聚体在PBS - 2）。 </li></ol></li><li>使活细胞钙黄绿素的积极和乙锭二聚体负，而死亡细胞钙黄绿素负和乙锭二聚体积极的，应调整仪器设置。 </li><li>图表和统计分析的酒吧可以在这些值可表示这些人口的百分比。 </li><li>测量不同的实验组。 </li></ol> 6。代表性的成果: 我们的研究结果表明，添加间质干细胞能改善缺血心肌细胞的生存。共聚焦显微镜图像显示，直接的细胞与细胞之间的相互作用，如部分膜连接或间碳纳米管，这可能发挥了重要作用，在观察的保护10。这些纳米管跨越几个细胞D的距离iameters，其直径是200至500纳米（白色箭头）。 <img src="/files/ftp_upload/3575/3575fig1.jpg" alt="图1" /> 图1。碳纳米管形成细胞之间。Vybrant DIO标记cardiomyoblasts Vybrant没有标记的骨髓基质干细胞之间的碳纳米管形成（白色箭头）。 流式细胞仪分析表明尚存的人口和坏死cardiomyoblasts和间质干细胞。有趣的是，我们也观察到了第三，"受伤"cardiomyoblast的细胞群，其中包含钙黄绿素，但也包含指标的死细胞，乙锭二聚体（图2）。 <img src="/files/ftp_upload/3575/3575fig2.jpg" alt="图2" /> 图2。代表点情节模拟缺血后不同的细胞群 。 SHO多次实验的评价周三，除了间质干细胞缺血H9C2 cardiomyoblast增加显著的活细胞的比例相比，单独培养缺血后心脏肌细胞（图3）。 <img src="/files/ftp_upload/3575/3575fig3.jpg" alt="图3" /> 图3。相比，控制（控制与红外模型:90.36 ± 2.60比10.52 ± 0.48，*** P&gt; 0.001）模拟缺血后，除了增加后，干细胞治疗缺血cardiomyoblasts对活细胞的百分比显着减少间质干细胞（红外模型与红外+骨髓基质细胞:10.52 ± 0.48与25.21 ± 2.13，*** P&gt; 0.001）。数据代表平均值± SEM。统计数据进行了分析，Tukey多重比较事后检验采用单向方差分析。

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没有利益冲突的声明。

证明协议是在体外培养的方法干细胞治疗心肌梗死，这种模式的优点和缺点，更复杂的问题。显然，这不能反映复杂的（如免疫）期间和之后，心肌梗死发生的事件，但可以集中精力对缺血细胞的补充细胞的直接影响。 H9C2 cardiomyoblasts模拟缺血的影响，高度依赖OGD时间，通过对所使用的细胞数量和O 2浓度。一是必须准确地调整这些参数，并发现某些细胞类型的最佳条件，才能有一个标准?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>钙黄绿素- AM </td><td>分子探针</td><td> L3224，C3099 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr><tr><td>乙锭二聚体2 </td><td>分子探针</td><td> L3224，E3599 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr><tr><td> Vybrant DID </td><td>分子探针</td><td> V22887 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr></tbody></table> 表1。试剂。 <table border="1"><tbody><tr><td> 设备名称 </td><td> 公司 </td><td> 评论（可选） </td></tr><tr><td>蔡司显微镜PeCon细胞孵化体系</td><td> PeCon有限公司</td><td> <a href="http://www.pecon.biz/">www.pecon.biz/</a> </td></tr></tbody></table> 表2。设备。

stem cell transplantation in an in vitro simulated ischemia/reperfusion model

干细胞移植协议是1,2,3到临床实践发现他们的方式。获得更好的效果，使协议更强大，并寻找新植入的细胞来源，是在最近的研究 4,5的重点。调查的细胞疗法的有效性是不是一件容易的事，需要新的工具，以探讨在治疗过程中所涉及的机制6。我们设计了一个缺血/再灌注损伤的实验协议，以便在缺血/再灌注损伤，干细胞移植后的细胞连接，甚至亚细胞机制的观察和评估细胞疗法的疗效。 H9C2 cardiomyoblast细胞被放置到细胞培养板7,8。缺血葡萄糖与氧气低于0.5％的水平的培养基中150分钟的模拟。然后，正常的媒体和氧含量重新模拟再灌注。氧糖剥夺后，加入他们的文化派生的标记的人骨间质干细胞骨髓移植治疗受损的细胞。在临床试验中的首选，因为它们是方便，微创手术，易于扩展和自体骨髓间质干细胞。共培养24小时后，细胞染色与钙黄绿素和乙锭-二聚体区分活细胞和死细胞。此设置允许我们调查的使用共焦荧光显微镜间的连接和量化缺血细胞流式细胞仪的存活率。共聚焦显微镜显示，如细胞融合和间碳纳米管形成的两个细胞群的相互作用。流式细胞仪分析显示，3受损的细胞，可以绘制在图表上，并进行统计学分析集群。这些人群可以单独进行调查，并根据这些数据得出的结论可以在模拟的有效性的治疗方法。

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Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

我们演示了如何建立一个<em&gt;在体外</em&gt;缺血/再灌注模型，以及如何评价干细胞治疗缺血心肌细胞的影响。

在干细胞移植在体外模拟缺血/再灌注模型

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

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Medicine

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

14.0K 次观看.  Semmelweis University. 我们演示了如何建立一个在体外缺血/再灌注模型，以及如何评价干细胞治疗缺血心肌细胞的影响。

视频: Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

科研

医学

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

急性肾损伤和伤后纤维化小鼠缺血再灌注模型

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

在脊髓损伤的实验挫伤型号神经干细胞移植

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

缺氧缺糖再给氧作为<em&gt;在体外</em&gt;缺血再灌注损伤模型的研究血脑屏障功能障碍

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

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 ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

肠缺血再灌注损伤的小鼠模型

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

创作和移植的糖尿病伤口愈合模型中脂肪源性干细胞 (ASC) 表

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

脑缺血再灌注的双血管闭塞小鼠模型

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

重建显微外科缺血再灌注损伤研究的临床前大鼠模型

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...