这项工作是支持的卡罗林斯卡研究所的KID资金，瑞典辐射保护局，卡罗林斯卡医学院眼科研究基金会，Konung古斯塔夫枪OCH贝蒂尔Stohnes Stiftelse，圣Erik的眼科医院研究基金会，Ögonfonden:秒OCH Drottning Viktorias Frimurarstiftelse。

1。暴露于紫外线辐射<ol><li>前15分钟的曝光，麻醉一个雌性Sprague-Dawley大鼠用90毫克/千克Ketalar（氯胺酮）和10毫克/公斤Rompun（甲苯噻嗪）通过腹膜内注射的混合物。 </li><li>将动物在大鼠阻挡，并拧紧而不会造成一个中继线挤压13，直到固定大鼠的皮带。 </li><li>灌输Mydriacyl（托），10毫克/毫升，在两只眼睛的大鼠诱导散瞳。 </li><li>将动物，使一只眼睛被靠住一个狭窄的光束的紫外线辐射在300 nm 14与10nm的半最大值全宽和屏蔽对侧眼用黑胶带。 </li><li>公开大鼠单方面亚阈值剂量的1千焦/米2 UVR-B在300 nm为15分钟，15。 </li></ol> 2。解剖<ol><li>预先确定的暴露后的时间间隔（1，5，24和120小时）后，牺牲的6周龄大鼠（重量&lt;200克）的碳水化合物Ň二氧化碳窒息和颈椎脱位。 </li><li>摘除细胞核的眼睛。此后，把眼睛角膜下，后侧。然后，冲头通过施加的27口径，插管和推切向巩膜，以避免损坏，这是非常接近的巩膜透镜，一个最小的孔。然后用一对眼科手术剪刀切圆周背后的后部巩膜缘，直到可以升空。然后，用钝头弯钳提起的镜头。 </li><li>在显微镜下从透镜赤道移除残余的睫状体，保持透镜的平衡盐溶液中不再比5-10分钟。 </li></ol> 3。定量RT-PCR <ol><li>将镜头到350微升RA1裂解缓冲液（NucleoSpin RNA总RNA提取试剂盒II）和3.5μLβ-巯基乙醇2 ml Eppendorf管中的混合。 </li><li>在此混合，在室温下的30分钟期间保持镜头。 </li><li>均质的透镜与只有硬核的镜头的针，直到剩下从透镜（皮层和胶囊的镜头的裂解缓冲液中裂解）。从组合中移除的核心。 </li><li>店内样品立即在-70°C。 </li><li>解冻的样品，并按照协议"总RNA纯化培养的细胞和组织"（NucleoSpin RNA总RNA提取试剂盒II）。 </li><li>专区RNA样品在-70℃下</li><li>要验证足够去除DNA，从每个样品中，运行一个PCR在以下条件下（第1步:95℃2分钟;步骤2中进行40个循环:95℃20秒，55℃下持续20秒，72° ℃20秒;步骤3:72℃，7分钟）与p53 DNA的特异性引物，正向5&#39;-ACCCTCTGACCTTTTTCCCA-3&#39;和逆5&#39;-TGCTGGGATCTTAGGCACTC-3&#39;和Taq DNA的聚合酶（dNTPack）的，根据制造协议。预期的PCR产物为243个碱基对。 </li><li>运行的1.5％琼脂糖凝胶电泳以检测DNA中的243个碱基对的特异性PCR产物。为了准备在TBE缓冲液中的1.5％琼脂糖凝胶3.75克琼脂糖，TBE缓冲液在250毫升的解决这个问题。加入溴化乙锭（0.5毫克/毫升）500微升在500毫升1.5％的琼脂糖凝胶溶液。加热该混合物，在一个微波欧文和搅拌解决琼脂糖。 </li></ol>样品没有发现任何DNA特异的PCR产物在1.5％的琼脂糖凝胶电泳。 <ol start="9"><li> RNA样品（1微升），测量的浓度中的NanoDrop ND-1000分光光度计在260 nm（RNA）和280 nm（蛋白质）的样品的吸光度的比例。如果RNA蛋白质的RNA样品的吸光度的比率是2.0或以上，则RNA样品是纯的。如果RNA样品的吸光度的比例低于2.0，重做RNA纯化步骤3.5。 </li><li>以对应的1微克RNA样品的体积，并合成cDNA后，协议第一链cDNA合成试剂盒（ 第一链cDNA合成试剂盒用于RT-PCR）。 </li><li>在-20°C贮存cDNA样本（周期为1以上，储存在-70℃下）。 </li><li>运行实时定量PCR的iCycler MyiQ™实时单色实时PCR检测系统。的负载一式三份于96孔板与1μlcDNA样本，TaqMan基因表达的TaqMan母液，半胱天冬酶3的基因表达分析，根据生产指令。负载三份在另一96孔板中与1μlcDNA样品，TaqMan基因表达母液，TaqMan基因表达为18秒的测定，根据生产指令（TaqMan基因表达含量议定书）。在系列稀释cDNA的3个随机选择的非暴露镜头的一小部分。运行串行稀释液一起从样品中的96 - 孔板的cDNA。 </li><li>使用标准曲线法进行定量的结果。的周期的数量在阈值荧光用作MyiQ™实时软件中的测量。在相对集中的校准功能的阈值的标准曲线表示的周期数是ES建立起来，在每块板中的系列稀释液。 cDNA的未经处理的镜片样品用于校准。对每个样品的周期相关的阈值数的标准曲线，得到样品的cDNA的测量的相对浓度。最后，得到的靶基因的表达水平，由有关的内部控制，18S基因的cDNA的目标相对集中。 </li></ol> 4。免疫组化染色<ol><li>固定术<ol><li>解剖的眼睛在PBS（磷酸盐缓冲液，pH 7.4）中。 </li><li>将眼球的管中填充用4％的多聚甲醛（PFA）在冰冷的20分钟。准备PFA 4％的前一天。储存于4℃直至使用。 PFA是新鲜的大约一个星期。 </li><li>微量移除PFA。然后，20分钟，用冰冷的PBS洗涤。 </li><li>把的眼睛蔗糖的30％在4°C过夜。 </li><li>把眼睛中充满了最佳切削温度（OCT）介质一杯（组织-TEK）。在一个合适的位置，切片放在眼。 </li><li>干冰冻结OCT介质。 </li><li>储存于-70℃直至使用。 </li></ol></li><li>切片<ol><li>冰冻切片在适当的平面上放置的眼睛。 </li><li>切三5μm厚的矢状面中的部分从每个透镜的中心部分。放弃至少6节之间连续的部分，以免弄脏相同的细胞核中不同的部分。 </li><li>一旦部分已被切断，轻轻地放在一张幻灯片，在它的上面。本节应坚持的幻灯片。 </li><li>离开幻灯片，空气干燥，使用前。幻灯片可以储存在-20℃，直到需要。 </li></ol></li><li>荧光免疫组化<ol><li>让样品达到室温（5-10分钟）。 </li><li>绘制一个PAP笔（Invitrogen公司）的标本与周围的边缘。 </li><li>让边境干燥的一段时间（10分钟）。 </li><li>标签的显微镜幻灯片。 </li><li>补充水分，1×PBS 15分钟。 </li><li>通透标准块溶液中30分钟。 </li><li>加入一抗（兔多克隆抗体，切割的caspase-3抗体Asp175 966​​1;细胞信号技术公司）稀释1:3,000标准块的解决方案。 </li><li>储存在湿盒在4°C过夜。 </li><li>通过移液（3×5分钟），或通过浸渍在一个大的浴（&gt; 15分钟，1-2的变化），在PBS中清洗。 </li><li>添加在标准块溶液1:300稀释的第二抗体（抗兔第二抗体与一个特定的吸收/发射光谱）。 </li><li>通过移液（3×5分钟），或通过浸渍在一个大的浴（&gt; 15分钟，1-2的变化），在PBS中清洗。 </li><li>擦拭掉PAP笔涂片。 </li><li>存储在一个加湿室中在室温搅拌3小时。避免暴露在光线下。 </li><li>添加一个的Vectashield下降，并在幻灯片上放置盖玻片。避免气泡。 </li><li>让Vectashield变硬（〜20分钟）。 </li><li>保持第In是黑暗的，直到分析。 </li><li>所有的控制部的处理的情况下的初级抗体。 </li><li>查找的结果，在荧光显微镜下在几个小时内。 </li><li>计数上皮细胞的细胞核从一个侧面核弓对方核弓的每一个镜头。应用标准的蓝色滤色器看到所有镜头上皮细胞核在蓝色和一个标准的过滤器适合的二次抗体的发射绿色看到的caspase-3阳性细胞核。 </li><li>记录全部晶状体上皮细胞核和阳性细胞核的数目的数目。计数细胞3次的每个部分。 </li></ol></li></ol>

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没有利益冲突的声明。

<html>本文介绍的方法，使学习在UVR-B引起的白内障发生的分子事件。 考虑，大多数信息可用于在体内的紫外线辐射性白内障是来自实验白化只SD大鼠7日，16日，17日，18日，19后，我们决定在目前的研究中使用的白化SD大鼠。年龄的影响是6周大的。的性别是女性，因为在对男性相比，女性较少有过敏尿。此外，有没有性别差异的严重程度的紫外线辐射性白内障...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> Ketalar </td><td>辉瑞公司</td><td> 150086 </td><td> 50毫克/毫升</td></tr><tr><td> Rompun </td><td>拜耳</td><td> 022545 </td><td> 20毫克/毫升</td></tr><tr><td>眼膏单</td><td> Apoteket，瑞典</td><td> 336164 </td><td> 5克</td></tr><tr><td> Mydriacyl </td><td>爱尔康</td><td> 00352 </td><td> 10毫克/毫升</td></tr><tr><td>平衡盐溶液</td><td>爱尔康</td><td> 0007950055 </td><td></td></tr><tr><td> 3.5 ULβ-巯基乙醇</td><td></td><td></td><td></td></tr><tr><td> NucleoSpin RNA II总RNA提取试剂盒</td><td> MACHEREY-NAGEL GMBH＆CO，德国Düren </td><td> 740955.50 </td><td></td></tr><tr><td> p53的DNA特异引物</td><td> biomers.net有限公司</td><td>订制</td><td></td></tr><tr><td> Taq DNA聚合酶，dNTPack </td><td>罗氏公司</td><td> 04 728 866 001 </td><td></td></tr><tr><td>琼脂糖</td><td>西格玛</td><td> A5093 </td><td></td></tr><tr><td>溴化乙锭溶液0.5毫克/毫升</td><td>西格玛</td><td> E1385 </td><td></td></tr><tr><td>纳米滴ND-1000分光光度计</td><td>的NanoDrop产品</td><td></td><td></td></tr><tr><td> 第一链cDNA合成试剂盒用于RT-PCR（AMV） </td><td>罗氏诊断公司</td><td> 11 483 188 001 </td><td></td></tr><tr><td>单色的iCycler MyiQ™实时实时PCR检测系统</td><td> Bio-Rad公司实验室</td><td></td><td></td></tr> &lt;TR&gt; <td> 96 - 孔板</td><td>萨尔斯塔特</td><td> 72.1979.202 </td><td></td></tr><tr><td> TaqMan基因表达预混</td><td>美国应用生物系统公司</td><td> 4369016 </td><td></td></tr><tr><td> caspase 3的TaqMan基因表达分析</td><td>美国应用生物系统公司</td><td> Rn00563902_m1 </td><td></td></tr><tr><td>对于18岁的TaqMan基因表达分析</td><td>美国应用生物系统公司</td><td> Hs99999901_s1 </td><td></td></tr><tr><td> MyiQ™实时软件</td><td> Bio-Rad公司实验室</td><td></td><td></td></tr><tr><td>劈开的caspase-3（Asp175） </td><td>细胞信号技术</td><td> 9661 </td><td></td></tr><tr><td>抗兔IgG </td><td> Abcam公司</td><td> Ab6798 </td><td></td></tr><tr><td>蔗糖</td><td> Sigma Aldrich公司</td><td> 84097 </td><td></td></tR&gt; <tr><td> Vectashield </td><td>矢量实验室</td><td></td><td></td></tr><tr><td>万能工具显微镜Axioplan 2影像</td><td>卡尔·蔡司</td><td></td><td></td></tr><tr><td>多聚甲醛</td><td> Sigma Aldrich公司</td><td> 441244 </td><td></td></tr><tr><td> TBE缓冲液10倍</td><td> Promega公司</td><td> V4251 </td><td></td></tr></tbody></table>

characterization of molecular mechanisms of in vivo uvr induced cataract

白内障是导致失明的世界1。世界卫生组织定义白内障混浊的眼睛，阻碍了光传输的镜头。白内障是一种多因素的疾病，与糖尿病，吸烟，紫外线辐射（UVR），酒精，电离辐射，类固醇和高血压有关。有强烈的实验2-4和流行病学证据5,6，紫外线辐射会导致白内障。我们开发了两种麻醉和非麻醉动物的紫外线B诱导白内障的动物模型。 唯一的治疗白内障的手术治疗，但这种治疗方法是无法访问的所有。据估计10年白内障的发病延迟可以减少白内障手术的需要的50％9。为了延缓白内障的发生率，它是需要了解白内障的形成机制和有效的预防地层几种学习策略。白内障发展的机制之一，细胞凋亡起着至关重要的作用在启动白内障在人类和动物的10。最近我们的重点是在镜头凋亡的机制白内障的发展8,11,12。据预测，更好地了解紫外线辐射的影响，对细胞凋亡通路的发现新的药物，以防止白内障的可能性。 在这篇文章中，我们将介绍如何白内障可以通过实验诱导体内暴露于UVR-B。进一步的RT-PCR和免疫组化被作为工具来研究UVR-B诱发白内障的分子机制。

测量中的变化的各种来源，估计与方差分析，并发现，考虑三个测量每只动物的方差用于测量的时间是在15％的动物的顺序。因此，考虑到整个透镜分析，这是不可能的，以增加的精度。正交试验阐明了统计上显着的对比度，120小时之间延迟的时间间隔与较短的延迟时间间隔的caspase-3的消息。 在体内紫外线辐射暴露诱导的caspase-3的表达。 <img alt="?...

Watch this Scientific Journal Video about Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract at JoVE.com

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

白内障是导致失明的世界。太阳紫外线辐射（UVR）是白内障发展的主要危险因素。远紫外线B诱导白内障动物模型的开发。在这篇文章中，我们描述了白内障的形成进行调查:暴露于紫外线辐射，定量RT-PCR和免疫组化的方法。

表征的分子机制<em在体内</em&gt;紫外线辐射诱发白内障

Cataract is the leading cause of blindness in the world 1. The World Health Organization defines cataract as a clouding of the lens of the eye which ...

characterization-molecular-mechanisms-vivo-uvr-induced

Research

JoVE Journal

Medicine

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

9.8K 次观看.  Karolinska Institutet. 白内障是导致失明的世界。太阳紫外线辐射（UVR）是白内障发展的主要危险因素。远紫外线B诱导白内障动物模型的开发。在这篇文章中，我们描述了白内障的形成进行调查:暴露于紫外线辐射，定量RT-PCR和免疫组化的方法。

视频: Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

建立临床相关<em&gt;离体</em&gt;调查上皮创伤修复的微环境本地人模拟白内障手术模型

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

科研

发育生物学

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented with increasing depth, the incidence rate of cutaneous melanoma has shown a rapid and continuous increase during recent decades. In order to find effective preventative methods, it is important to understand the early steps of melanoma initiation in the skin. Previous data has demonstrated that follicular melanocyte stem cells (MCSCs) in the adult skin tissues can act as melanoma cells of origin when expressing oncogenic mutations and genetic alterations. Tumorigenesis arising from melanoma-prone MCSCs can be induced when MCSCs transition from a quiescent to active state. This transition in melanoma-prone MCSCs can be promoted by the modulation of either hair follicle stem cells&#39; activity state or through extrinsic environmental factors such as ultraviolet-B (UV-B). These factors can be artificially manipulated in the laboratory by chemical depilation, which causes transition of hair follicle stem cells and MCSCs from a quiescent to active state, and by UV-B exposure using a benchtop light. These methods provide successful spatial and temporal control of cutaneous melanoma initiation in the murine dorsal skin. Therefore, these in vivo model systems will be valuable to define the early steps of cutaneous melanoma initiation and could be used to test potential methods for tumor prevention.

The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

突变黑色素细胞干细胞引发小鼠黑色素瘤的时空控制

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...

癌症研究

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

表皮黑色素和防止晒斑的人性化小鼠模型的诱导药物

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

医学

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal abnormalities. In addition, pathological myopia is a common irreversible cause of visual impairment and blindness1-3. Our study demonstrates the effect of scleral crosslinking using riboflavin and ultraviolet-A radiation on the development of axial myopia in a rabbit model. The axial length of the eyeball was measured by A-scan ultrasound in New Zealand white rabbits aged 13 days (male and female). The eye then underwent 360&#176; conjunctival peritomy with scleral crosslinking, followed by tarsorrhaphy. Axial elongation was induced in 13 day-old New Zealand rabbits by suturing their right eye eyelids (tarsorrhaphy). The eyes were divided into quadrants, and every quadrant had two scleral irradiation zones, each with an area of 0.2 cm&#178; and a radius of 4 mm. Crosslinking was performed by dropping 0.1% dextran-free riboflavin-5-phosphate onto the irradiation zones 20 sec before ultraviolet-A irradiation and every 20 sec during the 200 sec irradiation time. UVA radiation (370 nm) was applied perpendicular to the sclera at 57 mW/cm&#178; (total UVA light dose, 57 J/cm&#178;). Tarsorrhaphies were removed on day 55, followed by repeated axial length measurements. This study demonstrates that scleral crosslinking with riboflavin and ultraviolet-A radiation effectively prevents occlusion-induced axial elongation in a rabbit model.

We demonstrate the effect of scleral crosslinking with riboflavin and UVA on an axial elongation rabbit eye. Axial elongation was induced in 13 day-old New Zealand rabbits (male and female) by suturing their right eye eyelids (tarsorrhaphy).

巩膜交联兔模型使用核黄素和紫外辐射的轴性近视防治

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal ...

The Lambda Select cII Mutation Detection System

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic mice and the Lambda (&#955;) Select cII Mutation Detection System have been employed for mutagenicity experiments by many research groups worldwide. Here, we describe a detailed protocol for the Lambda Select cII mutation assay, which can be applied to cultured cells of transgenic mice/rats or the corresponding animals treated with a chemical/physical agent of interest. The protocol consists of the following steps: (1) isolation of genomic DNA from the cells or organs/tissues of transgenic animals treated in vitro or in vivo, respectively, with a test compound; (2) recovery of the lambda shuttle vector carrying a mutational reporter gene (i.e., cII transgene) from the genomic DNA; (3) packaging of the rescued vectors into infectious bacteriophages; (4) infecting a host bacteria and culturing under selective conditions to allow propagation of the induced cII mutations; and (5) scoring the cII-mutants and DNA sequence analysis to determine the cII mutant frequency and mutation spectrum, respectively.

The Lambda Select cII Mutation Detection System

We describe a detailed protocol for the Lambda Select cII mutation assay in cultured cells of transgenic rodents or the corresponding animals treated with a chemical/physical agent of interest. This approach has been widely used for mutagenicity testing of carcinogens in mammalian cells.

Lambda 选择cII突变检测系统

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of ...

免疫与感染

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

生物学

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H2O2), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1&#946;, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-&#945;) assay, and cells were evaluated for viability using fluorescent dyes. 
The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H2O2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line.

确定紫外线辐射和化学物质对原代和永生化的人角膜上皮细胞的毒性

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...

Organ Culture System for Assessing the Toxicity of Intraocular Treatment Excipients and Pharmaceuticals

As the leading cause of blindness, cataracts are a significant burden for the tens of millions of people affected globally by this condition. Chemical exposures, among other environmental factors, are an established cause of cataracts. Ocular toxicity testing can assess whether pharmaceuticals and their components may contribute to lens damage that may lead to cataracts or aid the treatment of cataracts. 
In vitro studies and in vivo animal testing can be used for assessing the safety of chemicals prior to clinical studies. The Draize test-the current in vivo standard for ocular toxicity and irritancy testing-has been criticized for lack of sensitivity and objective measurements of determining ocular toxicity. In vitro cell-based assays are limited as cell cultures cannot appropriately model an intact functional lens. 
The method described here is a sensitive in vitro alternative to animal testing, designed to evaluate the response of the intact bovine lens to treatment at both the cellular activity level and for overall refractive performance. The non-toxic reagent resazurin is metabolized in proportion to the level of cell activity. The lens laser-scanner assay measures the ability of the lens to refract incident beams of light to a single point with minimal error, directly relevant to its natural function. The method may be used to determine both acute and delayed changes in the lens, as well as the recovery of the lens from chemical or environmental exposures.

The goal of this protocol is to evaluate changes in metabolic activity and refractive function of the lens in response to experimental treatment.

As the leading cause of blindness, cataracts are a significant burden for the tens of millions of people affected globally by this condition. Chemical ...

<meta charset="utf8"></head><body>通过 LEC 提取在小鼠模型中复制白内障手术技术

Modeling Cataract Surgery in Mice

Cataract surgery (CS) is an effective treatment for cataracts, a major cause of visual disability worldwide. However, CS leads to ocular inflammation, and in the long term, it can result in posterior capsular opacification (PCO) and/or lens dislocation driven by the post-surgical overgrowth of lens epithelial cells (LECs) and their conversion to myofibroblasts and/or aberrant fiber cells. However, the molecular mechanisms by which CS results in inflammation and PCO are still obscure because most in vitro models do not recapitulate the wound healing response of LECs seen in vivo, while traditional animal models of cataract surgery, such as rabbits, do not allow the genetic manipulation of gene expression to test mechanisms. Recently, our laboratory and others have successfully used genetically modified mice to study the molecular mechanisms that drive the induction of proinflammatory signaling and LEC epithelial to mesenchymal transition, leading to new insight into PCO pathogenesis. Here, we report the established protocol for modeling cataract surgery in mice, which allows for robust transcriptional profiling of the response of LECs to lens fiber cell removal via RNAseq, the evaluation of protein expression by semi-quantitative immunofluorescence, and the use of modern mouse genetics tools to test the function of genes that are hypothesized to participate in the pathogenesis of acute sequelae like inflammation as well as the later conversion of LECs to myofibroblasts and/or aberrant lens fiber cells.

<meta charset="utf8"></head><body>作者聚焦：揭示白内障手术后 PCO 和纤维化的分子机制

This method models cataract surgery in vivo by removing lens fiber cells from adult mice and leaving behind the capsular bag with attached lens epithelial cells (LECs). The injury response is then assessed at various times post-surgery using molecular and morphological criteria.

小鼠白内障手术建模

Cataract surgery (CS) is an effective treatment for cataracts, a major cause of visual disability worldwide. However, CS leads to ocular inflammation, and ...

表征的分子机制

摘要

探索更多视频

此视频中的章节

建立临床相关

突变黑色素细胞干细胞引发小鼠黑色素瘤的时空控制

表皮黑色素和防止晒斑的人性化小鼠模型的诱导药物

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C）损伤的反应

巩膜交联兔模型使用核黄素和紫外辐射的轴性近视防治

Lambda 选择cII突变检测系统

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

确定紫外线辐射和化学物质对原代和永生化的人角膜上皮细胞的毒性

Organ Culture System for Assessing the Toxicity of Intraocular Treatment Excipients and Pharmaceuticals

小鼠白内障手术建模

表征的分子机制

摘要

探索更多视频

此视频中的章节

建立临床相关

突变黑色素细胞干细胞引发小鼠黑色素瘤的时空控制

表皮黑色素和防止晒斑的人性化小鼠模型的诱导药物

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

巩膜交联兔模型使用核黄素和紫外辐射的轴性近视防治

Lambda 选择cII突变检测系统

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

确定紫外线辐射和化学物质对原代和永生化的人角膜上皮细胞的毒性

Organ Culture System for Assessing the Toxicity of Intraocular Treatment Excipients and Pharmaceuticals

小鼠白内障手术建模

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C）损伤的反应