该项目的筹资提供的研究和视网膜研究基金会的克莱顿基金会。

1。视网膜母细胞瘤tumorspheres的制备<ol><li>视网膜母细胞瘤肿瘤样本下取得的IRB批准的协议。 </li><li>准备好新鲜的干细胞媒体（Neurobasal的一个媒体，1X的B - 27补充零下20毫微克/毫升表皮生长因子，维生素A，1X非必需氨基酸溶液，1X L -谷氨酰胺，10纳克/毫升碱性成纤维细胞生长因子）。 </li><li>在一个无菌，组织使用手术刀文化的跨领域技术处理的6孔板，机械分解的肿瘤组织。立即加入100μL定义的干细胞媒体组织和肉末组织使用的手术刀轻轻蔓延。添加5毫升的定义干细胞媒体向好。 </li><li>在37℃，5％的CO 2在培养箱，每天检查。有些tumorspheres应该从中断的肿瘤组织几乎立即出现超过2-4天的过程中越来越多。 </li><li>每周5分钟，离心tumorspheres 300xg，悬浮在新鲜配制的干细胞媒体，和吸管达10倍，破坏较大，中央坏死领域，并允许新的，健康的领域，形成。 </li></ol> 2。注射肿瘤细胞的制备<ol><li>如果培养tumorspheres，轻轻吸管领域注入15 m​​L锥形管。轻轻吸管向上和向下与血清吸管扰乱领域的10-15倍。 </li><li>细胞被注入到一个新的管使用一个hemacytometer，等分至少50,000元注射细胞计数。准备尽可能多的基础上可用的细胞数量的注射。 </li><li>在300xg离心5分钟。仔细吸出上清液于5 mL PBS重悬。重复离心和愿望，并在每次注射10μL的PBS重悬。 </li></ol> 3。动物的制备<ol><li>使用胰岛素注射器，管理的啮齿类动物麻醉鸡尾酒的腹腔注射（氯胺酮37.6毫克/毫升，甲苯噻嗪1.92毫克/毫升，acepromazine 0.38毫克/毫升），每克体重1μL（GBW）。等待5分钟的动物成为镇静剂。检查心跳和一个加热垫放在动物。保持在所有时间，直到恢复加热垫的动物。 </li><li>镇静后，管理1右眼丙美卡因盐酸下降。更换加热垫的动物，并等待1分钟。 </li><li>管理福林盐酸右眼1滴。更换加热垫的动物，并等待5分钟。如果未发生瞳孔扩张，重管理的1滴，等待5分钟。 </li><li>监视运动或抽搐的迹象，这可能发生在30分钟后镇静，动物。在运动的最初迹象，管理1μL/ GBW盐酸氯胺酮（10毫克/毫升的PBS稀释），并等待5分钟。 </li></ol> 4。注射肿瘤细胞<ol><li>动物麻醉后，置于显微镜下，在其一侧与眼睛朝上的权利和头搁在纱布和中心，以获得红光反射的右眼眼底（瞳孔应该在这个时候扩张），当通过显微镜观察。 </li><li>撑起轻轻推，同时用两个手指在眼皮上和程序的其余部分持有这一立场稳步全球。 </li><li>使用连接到鲁尔锁注射器，无菌30号针头刺破全球横向通过结膜和巩膜玻璃体领域的角膜缘相邻（图1）​​，并通过入玻璃体腔脉络膜。从开放中取出针。 </li><li>汉密尔顿针用酒精棉签消毒。汉密尔顿注射器将细胞悬液，并绘制成针插入开幕，直到针头是通过在玻璃体视网膜附近的镜头背后的显微镜观察。第二而针稳定在这个位置上的人的帮助下，慢慢地压低柱塞。取出的针。如果有必要，用棉签从开放中吸收任何液体。 </li><li>轻轻松开眼睑的压力，关闭鼠标眼睑。 </li><li>管理的羟丙甲纤维素的1下降到每只眼睛和返回动物恢复加热垫。 </li></ol> 5。监测小鼠体内肿瘤的收获<ol><li>日报，研究注入了白瞳眼（白乳头状反射）和/或眼周，腹胀，通常2-4周注射后变得明显。 </li><li>一旦检测到肿瘤的生长，安乐死的动物根据机构指引。 </li><li>如果眼睛和肿瘤眼睑覆盖，使用的手术刀，使睑裂正交的两个切口，并解除皮瓣。 </li><li>使用可视化立体显微镜下，在角膜缘圆周的切口，去除角膜和晶状体，并认真剖析其他组织使用彧大部分的肿块zers。放置在无菌的RPMI - 1640媒体肿瘤。 </li><li>使用镊子，从插座上慢慢拉开放全球。为了确保一个附加的视神经，使肿瘤的侵袭，可以可视化，全球拉，直到神经暴露，并用剪刀切断神经，尽可能长时间。放置在10％福尔马林作病理检查的常规处理。 </li></ol> 6。代表结果: 视网膜母细胞瘤tumorspheres将开始从分类组织几乎会立即出现，因为它们是从肿块中解放出来。在2-4天之内，更多的tumorspheres将开始形成，并会增加大小。的领域往往是经常的，表现出一个明确的，二次膜周围聚集（图2）。 动物通常呈现4个星期内注射后（图3b），其次是扩大全球和腹胀注射5-8周后，对周围组织，随着肿瘤的增长与白瞳（图3c）。 <img src="/files/ftp_upload/2644/2644fig1.jpg" alt="图1"/> 图1。横断面图鼠标的眼睛突出在协议中所引用的功能。 <img src="/files/ftp_upload/2644/2644fig2.jpg" alt="图2"/> 图2。文化在体外培养的人视网膜母细胞瘤细 ​​胞成像）4X，和b）的10倍的目标放 ​​大倍率。视网膜母细胞瘤的原发肿瘤细胞产生一个普通的球体造型和清脆的外膜tumorspheres。比例尺代表）500微米， 和b）200微米。 <img src="/files/ftp_upload/2644/2644fig3.jpg" alt="图3"/> 白瞳在玻璃体腔内的肿块，C）一个大肿块 。RAG2 眼图 3 - / -小鼠显示一 ）正常功能，B）全球填补相关的眼周腹胀，眼内出血。

<ol>
	<li>Ch&#232;vez-Barrios, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+and+Nonmetastatic+Models+of+Retinoblastoma.">Metastatic and Nonmetastatic Models of Retinoblastoma.</a> Am. J. Pathol. 157, 1405-1412 (2000).</li><li>Suber, M. L., Hurwitz, M. Y., Ch&#232;vez-Barrios, P., Hurwitz, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+consequences+of+intraocular+administration+of+modified+adenoviral+vectors.">Immune consequences of intraocular administration of modified adenoviral vectors.</a> Hum. Gene Ther. 12, 833-838 (2001).</li><li>Mallam, J. N., Hurwitz, M. Y., Mahoney, T., Ch&#232;vez-Barrios, P., Hurwitz, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Gene+Transfer+into+Retinal+Cells+Using+Adenoviral+Vectors:+Dependence+on+Receptor+Expression.">Efficient Gene Transfer into Retinal Cells Using Adenoviral Vectors: Dependence on Receptor Expression.</a> Invest. Ophthalmol. Vis. Sci. 45, 1680-1687 (2004).</li></ol>

动物实验是由贝勒医学院医学IACUC委员会规定的准则和法规的规定执行。

本文所述的技术有利于他们的眼，玻璃体环境传播视网膜母细胞瘤肿瘤。在过去的眼内注射技术已用于创建肿瘤的视网膜母细胞瘤衍生的细胞系 ，1以及提供人工基因治疗的病毒载体2,3。这项技术目前已成功地用于生产人类视网膜母细胞瘤肿瘤细胞从原发肿瘤和注射的tumorspheres以及裸鼠移植瘤的串行传播直喷。肿瘤形成的明显证据（通常为白瞳症）是4个星期内，通常首先指出，在5-8周后，眼内出血及全球和/或组织腹胀周围的轨道发展。少数注入小鼠体内注入眼伤害，导致眼睑永久封闭。在这种情况下，腹胀是肿瘤形成的唯一标志。 建立小鼠移植瘤肿瘤尚未成功，所有的人类视网膜母细胞瘤瘤，虽然建立的裸鼠移植瘤的传播是非常成功的。这一观察结果表明，原发肿瘤，如分化或其他一些未知因素的侵袭和水平，某些特性可能会影响这些肿瘤的能力，坚持在小鼠眼部环境。 可以从一个人的视网膜母细胞瘤瘤收购的组织是相当小，并在体外培养的人视网膜母细胞瘤细 ​​胞，如寿命有限，失去坚实的肿瘤组织学和细胞表型的变化能力有很大的局限性。该协议提供了一种比较简单的方法传播人类肿瘤和建立人类疾病的小鼠模型。这使得进一步在体外和体内的视网膜母细胞瘤和更长的维护病人的肿瘤外的生物学实验。

<table border="1"><tbody><tr><th>名称</th><th>公司</th><th>目录＃ </th><th>评论</th></tr><tr><td>去氧肾上腺素盐酸2.5％ </td><td>博士伦</td><td> 053-11 </td><td>眼科解决方案</td></tr><tr><td> 30 - GA针</td><td>屋宇署</td><td> 305128 </td><td>定期斜角</td></tr><tr><td> 10毫升的鲁尔锁注射器</td><td>屋宇署</td><td> 309604 </td><td></td></tr><tr><td> 3/10-cc胰岛素注射器</td><td>屋宇署</td><td> 328431 </td><td></td></tr><tr><td>酒精棉签</td><td>屋宇署</td><td> 326895 </td><td></td></tr><tr><td> 6孔板，组织培养处理</td><td>屋宇署</td><td> 353046 </td><td></td></tr><tr><td>盐酸丙美卡因0.5％ </td><td>巴特勒热源</td><td> 017239 </td><td>眼科解决方案</td></tr><tr><td>氯胺酮盐酸100mg/mL </td><td>道奇堡</td><td> 4402A </td><td></td></tr><tr><td> 10μL汉密尔顿注射器</td><td>汉密尔顿公司</td><td> 7648-01 </td><td></td></tr><tr><td> 32 - GA汉密尔顿针</td><td>汉密尔顿公司</td><td> 7803-04 </td><td>自定义长度 - 0.5" </td></tr><tr><td> Neurobasal的媒体</td><td> Invitrogen公司</td><td> 10888-022 </td><td></td></tr><tr><td> B - 27补充减去维生素A，50X </td><td> Invitrogen公司</td><td> 12587-010 </td><td></td></tr><tr><td> RPMI - 1640媒体</td><td> Mediatech </td><td> 10 - 040 - CV </td><td></td></tr><tr><td>非必需氨基酸溶液，100X </td><td> Mediatech </td><td> 25 - 025 - CI </td><td></td></tr><tr><td> L -谷氨酰胺，100X </td><td> Mediatech </td><td> 25 - 005 - CI </td><td></td></tr><tr><td>一次性＃11手术刀</td><td> Miltex </td><td> 4-411 </td><td></td></tr><tr><td>鼠患麻醉组合</td><td> N / A </td><td> N / A </td><td>在药剂配方（氯胺酮37.6毫克/毫升，甲苯噻嗪1.92毫克/毫升，acepromazine 0.38毫克/毫升） </td></tr><tr><td>重组人表皮生长因子（EGF） </td><td>干细胞技术</td><td> 02633 </td><td>在10微克/毫升原液的重组</td></tr><tr><td>重组人碱性成纤维细胞生长因子（bFGF） </td><td>干细胞技术</td><td> 02634 </td><td>在10微克/毫升原液的重组</td></tr><tr><td> OMS - 75手术显微镜</td><td>拓普康医疗系统</td><td> OMS - 75 </td><td>这种模式已停产</td></tr><tr><td> 10％的福尔马林</td><td> VWR </td><td> 95042-908 </td><td></td></tr></tbody></table>

establishment and propagation of human retinoblastoma tumors in immune deficient mice

培养视网膜母细胞瘤肿瘤细胞在界定干细胞媒体产生可以种植和只是一个有限的时间内保持的小学tumorspheres。原发肿瘤相比，这些培养tumorspheres可能会出现明显不同的细胞表型。示范，培养的细胞形成新的肿瘤的能力是很重要的原发肿瘤的生物学特性，以确保培养的细胞模型。 在这里，我们目前为传播使用RAG2 体内的人类视网膜母细胞瘤的协议- / -免疫缺陷小鼠。低通道或从刚收获的人类的视网膜母细胞瘤直接注入小鼠眼睛的玻璃体腔内肿瘤细胞体外培养的人类视网膜母细胞瘤tumorspheres的形成2-4个星期内肿瘤。这些肿瘤可收获，要么进一步代入小鼠体内的眼睛，或在体外tumorspheres增长。传播已成功地进行了至少有三个通道从而确立了人类视网膜母细胞瘤组织继续为进一步的实验源。 韦斯利债券和拉丽塔瓦德瓦共同第一作者。

Watch this Scientific Journal Video about Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice at JoVE.com

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

描述的方法传播人类视网膜母细胞瘤小鼠的肿瘤。肿瘤细胞直接注入免疫缺陷小鼠的眼睛。继发性肿瘤已成功地建立了使用直接从人类肿瘤和培养tumorspheres收获细胞。

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

establishment-propagation-human-retinoblastoma-tumors-immune

Research

JoVE Journal

Medicine

11.7K 次观看.  Baylor College of Medicine. 描述的方法传播人类视网膜母细胞瘤小鼠的肿瘤。肿瘤细胞直接注入免疫缺陷小鼠的眼睛。继发性肿瘤已成功地建立了使用直接从人类肿瘤和培养tumorspheres收获细胞。

视频: Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

浸润性膀胱移行细胞癌中的完整的人MUC1转基因小鼠免疫诱导免疫治疗发展模型

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

科研

医学

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

股骨骨髓的愿望在活体小鼠

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

创建精确的解剖学和人脑肿瘤的重现性颅内移植瘤

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

从整体组织和活检建立人类上皮Enteroids和Colonoids的

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

在小鼠节段性股临界尺寸缺陷模型的建立接骨板稳定

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

小鼠加速动脉硬化的诱导:"电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

应用流式细胞仪对人体脂肪组织免疫细胞的表征

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.