作者要感谢MALINDA轻快的技术援助。我们也承认目前和过去​​资金来源:美国国家癌症研究所（R01 CA131075，R01 CA131075-02S1），温迪威尔案例癌症研究基金，马基癌症基金会，儿童奇迹网络和珍妮弗与大卫·狄更斯黑色素瘤研究基金会。

1。从左手石竹（Cohleus毛喉）植物的粗根提取物制剂毛喉素用于局部给药的<ol><li>协议对小鼠实验其次为动物的护理和使用道德行为的准则和批准的机构动物护理和使用委员会在肯塔基大学（协议＃00768M2004）。根提取物是由在40％重量/体积70％乙醇中的一个标准皮肤病学的基础上，30％的丙二醇。 </li><li>权衡将200g粗福斯克林根提取物，并将其转移到烧杯中。制成500毫升40％（重量/体积）溶液，重悬将200g粗福斯克林根提取物，加入大部分但不是全部车辆的体积（70％乙醇，30％丙二醇），并把该溶液至大约450毫升。 </li><li>搅拌一个小时在室温下。该解决方案将有点粘稠，可能需要手动搅拌以"升降机"提取到溶液中之前搅拌棒是能够接管​​。 </li><li>搅拌一小时后，混合物倒入量筒中，并使用车辆已被用于"漂洗"，是用来搅拌的浆料中的烧杯中（最大限度地从烧杯中恢复毛喉素），使体积至500ml 。 </li><li>将浆料转移至50ml聚丙烯离心管中。离心（1,500×g离心，室温，15分钟）使用台式离心机。在这一点上，不溶性材料将是相当密实，使上清液容易被倒出。 </li><li>通过0.22微米乙酸纤维素膜过滤该溶液，以从提取物中除去残留的不溶性物质。我们使用的瓶顶系统设计用于细胞培养，随着使用的预过滤器来与单元以防止从根提取物的不溶性组分的膜过早堵塞。当使大量的提取物，过滤大致100毫升在一个时间，改变了预过滤器的选择吐温每个添加量。 </li><li>当在室温下保存，提取物保持生物学活性高达长达一年。 </li></ol> 2。用于局部治疗制备C57BL / 6 K14-SCF小鼠<ol><li>电动剪切从动物取出背毛皮。简要麻醉动物吸入异氟醚，方便背部皮毛配备0.25mm的手术预备头（飞世尔科技）电热剪的剪切。最好只使用一种类型的麻醉（ 如氯胺酮/甲苯噻嗪），以尽量减少麻醉药过量的风险。饱和吸入室进行职业风险时，通风橱外使用，并提供未知量的麻醉剂的动物。理想的情况是一个精密蒸发器应该被使用。 </li><li>以去除残留的毛茬，善待动物用化学脱毛剂。管理麻醉的动物用腹腔注射氯胺酮40 mg / kg和甲苯噻嗪4毫克/千克</li&gt; <li>一旦动物有足够的麻醉（用脚趾捏来判断），适用的脱毛霜指尖大小的量，用戴手套的手指剪切背侧皮肤。擦眼霜渗入皮肤30-60秒或直到毛发可以清楚地在奶油被看作是它被搬来搬去。离开上霜只对需要的时间脱毛的最小量为长时间暴露引起的皮肤，表皮破裂和死亡的表皮完整性损失的化学燃烧。 </li><li>反复擦拭背部皮肤用清水浸泡过的纱布垫，直到所有的冰淇淋已被删除。用柔软的纸巾，并允许他们在一个温暖的僻静的位置（ 如干净的笼子放置在一个变暖垫）恢复干燥的动物。脱毛的动物，一个接一个，整个手术过程中密切监测。 </li></ol> 3。毛喉素或车辆控制的局部给药<ol><li>动物应该在一个时间进行处理1。与吸入麻醉简要通过将鼠标上的下一个形状吻合尼龙透气式过滤器的顶部ð异氟醚已被放置的异氟醚饱和的纸巾在通风橱中的异氟醚的饱和带盖透明玻璃广口瓶中。暴露的小鼠对异氟烷足够的时间，以抑制自发肌肉运动，但保持自主呼吸（通常是10-20秒）。留在异氟醚的动物过长会导致呼吸抑制而死亡。这是更好地犯错"去光"，不得不重新暴露鼠标简单地多异氟醚，而不是动物过度暴露于药物和死亡风险的一面。 </li><li>从异氟醚室中取出的动物，并放置在干净的吸水垫板凳。 </li><li>使用1,000微升微量配备一次性聚丙烯尖，制定400微升40％的粗提取物毛喉素（车辆控制动物将获得70％的乙醇，独自一人30％丙二醇）。 </li><li>提取物转印到背面的动物通过它滴落到皮肤上，然后，用移液管尖端的侧面，涂抹提取在背部皮肤，直到所有的皮肤已经被覆盖。有没有必要向吸干涂布后的皮肤上。 </li><li>返回鼠标笼子里，并仔细观察，直到它从麻醉中恢复。 </li><li>为了使非色素cAMP的作用不应该混淆紫外线敏感性试验，停止所有的局部治疗前2天的紫外线照射（颜料效果持续数天超越去年局部治疗）。 </li></ol> 4。皮肤颜色测量由反射比色法<ol><li>简要麻醉小鼠吸入异氟醚（见上文）。 </li><li>通过将便携式头所提供的色度计标准化白色表面校准美能达色差。 </li><li>将色度计平齐的便携式测量头与该动物的背部皮肤确保1cm 2的圆形aperture的完全压在皮肤上。采取在背部皮肤的不同区域的至少三个单独的测量。 </li><li>计算平均长*得分±每只动物和每个治疗组的SD。反射色度可以在任何时候在实验中进行。 </li></ol> 5。由"最小红斑剂量"（MED）的计算测定紫外灵敏度<ol><li>使用已预先用载体或如上述毛喉素的动物。麻醉动物腹腔注射氯胺酮和甲苯噻嗪（见上文）的标准混合物。 </li><li>准备一张紫外闭塞磁带MED测试。以产生在带的孔中，使用了重型打孔器以1厘米2的圆形开口（ 图2A和B）。具有在带一个定义尺寸和对称布置的孔便于照射后识别的皮肤变化。多年来在磁带每孔，应用一个小而易于拆卸件<html>磁带，可以在规定的时间紫外线照射过程中被移除，以允许不同的紫外线剂量给药。 </li><li>一旦动物有足够的镇静，将磁带上的背水面。眼部润滑剂应始终在麻醉下利用。 </li><li>打开UV光源包括两个西屋F15T8UV-B灯与313纳米的峰值输出和一系列的280-370纳米。允许通过紫外分光光度计用紫外线传感器测得的灯，以平衡到一个恒定的紫外线输出（通常需要几分钟，让灯预热）。 </li><li>根据如通过UV光度计测得的紫外线透射率，计算出的紫外线照射时间为每个所需的剂量。例如，我们的灯的紫外线输出的措施2.4毫瓦/厘米2。因此，施用5千焦耳/米2，皮肤将需要暴露在208秒（这是3分钟，28秒）的UVB辐射，计算如下: upload/50670/50670eq1.jpg"/&gt; </li><li>将动物镇静剂（每到位闭塞磁带）腹面向下，以确保即使紫外线照射。施用所选择的剂量的紫外线辐射，依次取出小闭塞胶带覆盖该孔以露出1厘米皮肤的2个区域，以正确的剂量的辐射。因此，使用上面的例子，如果40千焦耳/米2是实验中的最大剂量，则该动物将灯泡下为27分钟和47秒的总和皮肤中的40千焦耳/米2的条件就没有覆盖磁带的整个时间。但是，磁带上覆5千焦/米2的条件时，会出现208秒剩余曝光被删除。胶带去除时机应该做的，让每个条件同时结束。 </li><li>紫外线照射后，撕下胶带小心地从背部皮肤，注意不要撕裂突然或过度有力的运动的皮肤。将动物在温暖安静的地方，让麻醉恢复期。 </li><li>监测小鼠为24-48小时，以寻找红斑（发红）或水肿的离散区域（肿胀）对应于暴露于UV辐射的特定剂量的解剖部位。照相记录皮肤的发现。 </li><li> MED值对应于最小剂量的紫外线红斑和/或皮肤的整个暴露圆的水肿定义，导致炎症。需要注意的是皮肤的色素沉着可以挑战的决心MED，然而，红斑和水肿仍然可以大致准确地评估，这要部分归功于在磁带上的小孔中的紫外线照射过程中定义的形状。 </li></ol> 6。统计分析通过单因素方差分析和Bonferroni事后检验（图垫棱镜）。P值 &lt;0.05被认为是统计学显著分析老鼠的同伙之间的数据。

作者宣称，他们有没有竞争的财务权益。

使用皮肤白皙的人的动物模型，我们发现一个毛喉素富含粗根提取物的局部应用强劲，刺激黑色素的生成在皮肤变暗的表皮。表皮黑色素是依赖于干细胞因子在基底表皮的表达，如发生在人类皮肤，但不能在基因未修饰小鼠皮肤。基因未修饰小鼠的背部皮肤缺乏足够数量的滤泡黑色素细胞以赋予颜料在皮肤上。只在黑素细胞生长因子，如干细胞因子（kit配体）或肝细胞生长因子（HGF）的组成型表?...

黑色素瘤的发病率，皮肤癌的最致命的形式，也大幅增加，在过去几十年在美国，尤其是皮肤白皙的人。强烈的分子和流行病学证据牵连紫外线辐射为主要致病环境因素2-5。在阳光下暴晒和晒黑床使用的形式增加紫外线照射很可能是负责大部分的增加黑色素瘤的发病率6-7。黑色素瘤的风险似乎与晒伤8，尤其是那些生活在9-10月初特别的联系。晒伤的风险被链接不仅剂量和紫外线照射的强度，而且还通过影响紫外线辐射的皮肤反应遗传因素。皮肤色素沉着是一种UV灵敏度的最重要的决定因素，晒伤和癌症风险的风险。黑色素瘤约20倍更频繁地发生在浅肤色的人相比，皮肤黝黑的individuaLS 11-13。 黑色素，由黑素细胞在表皮中产生的颜料，是肤色的主要决定因素。黑色素有两个主要品种有:（1）真黑素，黑棕色/黑色颜料在吸收UV辐射的能量有效的，并且（2）褐黑素，红/金色颜料防止紫外线光子的穿透入皮肤效果较差。肤色，紫外线的敏感性和黑色素瘤的风险在很大程度上是由表皮黑色素含量14-15决定。在表皮中的黑色素多，越少的UV光子可以渗透进皮肤。因为真黑素的先天低层次的，皮肤白皙的人对紫外线辐射16-18急性和慢性影响更容易发生。 皮肤色素沉着，黑色素瘤的风险，并有能力"晒黑"的紫外线照射后，所有与关联的黑素皮质素受体​​1（MC1R）的信令能力，一个G S耦合的七次跨膜s对黑素细胞19-22 urface受体。当MC1R结合其同源的高亲和力配体，α-黑素细胞刺激激素（α-MSH），有活化腺苷酸环化酶的第 ​​二信使cAMP 23的制作中。紫外线照射后皮肤的正常生理反应包括表皮生产α-MSH的由角质形成细胞24-29。我们和其他人推测，角质形成细胞源性α-MSH结合MC1R对表皮黑素细胞，通过激活腺苷酸环化酶30的启动下游生产内cAMP第二信使。 cAMP水平控制黑色素细胞分化的许多方面，包括存活途径，DNA修复和颜料的合成。 MC1R信号和cAMP诱导清楚色素酶水平和黑色素的生产。当MC1R信号是否完好及黑素细胞内cAMP水平是强大的，真黑素的产生和皮肤变暗。但是，如果MC1R信号是有缺陷的，并胞浆内cAMP水平仍然很低，褐黑素产生，而不是1。真黑素合成可以通过药理学的提高cAMP水平1,14,31-35剂刺激。 由于MC1R蛋白是在人类36-46黑素瘤风险的主要调节剂，我们感兴趣的是其中MC1R保护皮肤免受紫外线诱导黑素细胞癌变的机制。作为我们研究的基础，我们产生了转基因MC1R基因变体小鼠模型在纯C57BL / 6遗传背景1。在这个模型中，干细胞因子（SCF）的组成型表达在基底表皮和表皮黑素细胞滤泡被保留在整个生命47的皮肤，而相比之下，在非转基因小鼠，其中黑素细胞定位于毛囊真皮。与K14-SCF基因掺入，表皮变得与颜料的特定黑色素着色特性动物1株。K14-SCF小鼠对C57BL / 6遗传背景与野生型MC1R信号已经乌黑的皮肤特点是非常高水平的真黑素色素。毫不奇怪，这些动物是高度抗紫外线。相比之下，基因匹配K14-SCF的C57BL / 6动物怀有一种突变无效的MC1R在表皮几乎没有黑色素。相反，这些" 延伸 "的动物（MC1R E / E）具有引起的褐黑素色素（ 图1A）沉积公平肤色和更为紫外线敏感48-49。 与化学特性，使渗透到皮肤药物化合物已被证明直接操作cAMP水平的表皮黑素细胞在皮肤有效地诱导黑色素的扩展名（MC1R E / E）K14-SCF动物模型。在这个模型中黑色素的上调已经Ř通过腺苷酸环化酶激活1以及磷酸二酯酶4抑制35 eported。在这篇文章中，我们展示了准备并在扩展名（MC1R E / E）K14-SCF的动物模型中，皮肤白皙紫外线敏感的人毛喉素局部应用。我们表明，该药物每日两次应用促进加速黑色素，即皮肤变黑是由于黑色素的表皮沉积而引起的表皮黑色素防止紫外线引起的晒伤通过"最小红斑剂量"（MED）48的测量值。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>Coleus Forskoli extract 20%</td><td>Buckton Scott USA Inc.</td><td>n/a</td><td>Princeton, NJ</td></tr><tr><td>Isothesia, Isoflurane , USP</td><td>Butler Schein</td><td>NCD 11695-6776-1</td><td>Dublin, OH, USA</td></tr><tr><td>Xylazine</td><td>Anased Injection</td><td>LA04612</td><td>Shenandoah, Iowa, USA</td></tr><tr><td>Ketamine HCl, USP</td><td>Putney</td><td>NDC 26637-411-01</td><td>St. Joseph, MO, USA</td></tr><tr><td>Ethanol</td><td>Decon Labs.</td><td>2705</td><td></td></tr><tr><td>Propylene glycol</td><td>Adesco</td><td>05751L</td><td>Solon, OH, USA</td></tr><tr><td>Depilatory cream, Nair</td><td>Church Dwight</td><td>JF-11 4381322</td><td>Priceton, NJ</td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>Germicidal Hg Lamp UV-B</td><td>Westinghouse</td><td>F15T8UV-B</td><td></td></tr><tr><td>Radiometer photometer</td><td>International light</td><td>1LT400A</td><td>Peabody, MA,USA</td></tr><tr><td>Chromameter</td><td>Konica Minolta</td><td>CR-400</td><td>Ramsey, NJ, USA</td></tr><tr><td>Data Processor for Chromameter CR-400</td><td>Konica Monilta</td><td>DR-400</td><td>Ramsey, NJ, USA</td></tr></tbody></table>

pharmacologic induction of epidermal melanin and protection against sunburn in a humanized mouse model

公平性皮肤，紫外灵敏度和皮肤癌的风险的所有关联与的黑皮质素-1受体的生理功能，黑素细胞的表面上发现了G S耦合信号蛋白。 MC1R刺激腺苷酸环化酶和cAMP的产生，这反过来，上调黑色素细胞黑色素的产生在皮肤上。为了研究由MC1R信令保护皮肤免受紫外线伤害皮肤的机理，本研究依赖于小鼠模型与基于干细胞因子（SCF）的表达表皮"人源化的皮肤"。K14-SCF基因小鼠黑素细胞保留在表皮，因此具有沉积黑色素在表皮的能力。在这个动物模型，在黑色的真黑素色素和UV保护型健壮的沉积野生型MC1R状态结果。与此相反，K14-SCF动物有缺陷的MC1R信令能力表现出红色/金色的色素沉着，很少真黑素在皮肤和一个对紫外线敏感的表型。理由是真黑色素的沉积可能是由局部用药，模仿MC1R信号增强，我们发现直接应用毛喉素提取物对MC1R基因缺陷的皮肤白皙的小鼠的皮肤造成了强大的真黑素诱导和防紫外线1。在这里，我们描述了制备和应用含毛喉素天然根提取物对K14-SCF皮肤白皙小鼠及报告，通过测定最小红斑量（MED）测定紫外灵敏度的方法的方法。使用该动物模型，能够研究的皮肤表皮的cAMP的诱导和黑化如何影响生理反应，以UV曝光。

上结合了K14-SCF基因所描述的（ 图1A）eumelanotic，pheomelanotic或无色素的背景中产生C57BL / 6小鼠。皮肤白皙的扩展同伙（MC1R E / E，酪氨酸+ / +）小鼠，每日两次剂量溶媒（70％乙醇，30％丙二醇）或40％的粗毛喉鞘蕊根提取物（每80微米的局部治疗剂量），连续5天（ 图2B）。对表皮色素沉着的局部治疗效果进行既通过目测和通过反射色度（ ?...

Watch this Scientific Journal Video about Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model at JoVE.com

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

表皮黑色素是由毛喉素局部应用在皮肤白皙的紫外线敏感的人小鼠模型诱导。 cAMP水平的皮肤和表皮发黑免受紫外线介导的炎症（晒斑）强烈保护如由最小红斑量（MED）法测定的药理操纵。

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 ...

pharmacologic-induction-epidermal-melanin-protection-against-sunburn

Research

JoVE Journal

Medicine

18.1K 次观看.  University of Kentucky College of Medicine. 表皮黑色素是由毛喉素局部应用在皮肤白皙的紫外线敏感的人小鼠模型诱导。 cAMP水平的皮肤和表皮发黑免受紫外线介导的炎症（晒斑）强烈保护如由最小红斑量（MED）法测定的药理操纵。 

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

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

低聚活性的体外定量

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

科研

生物化学

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

黑色素瘤患者衍生异种移植模型

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

癌症研究

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 ...

Chemical-Induced Skin Carcinogenesis Model Using Dimethylbenz[a]Anthracene and 12-O-Tetradecanoyl Phorbol-13-Acetate (DMBA-TPA)

Cancer is one of the most devastating human diseases. Experimental cancer models are important to gain insight into the complex interplay of different cell types and genes in promoting tumor progression and to provide a platform for testing the efficacy of different therapeutic approaches. One of the most commonly used experimental inflammatory cancer models is the DMBA-TPA two-stage skin carcinogenesis model. Tumor formation is induced in this model by the topical application of two different chemicals, 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoyl phorbol-13-acetate (TPA), that together cause papilloma formation in the skin. As the primary outcome is papilloma formation in the skin, the model is an ideal, reliable, and reproducible way to address both tumor initiation (tumor-free survival) and tumor progression (number and size of visible tumors). The effects of the DMBA-TPA treatment are transmitted via an inflammatory mechanism, which makes this model especially suitable for studying the role of the immune system in tumor formation. However, this model is restricted to the skin and other surfaces where the chemicals can be applied on. A detailed protocol is provided in this article to use the model successfully.

Chemical-Induced Skin Carcinogenesis Model Using Dimethylbenz[a]Anthracene and 12-O-Tetradecanoyl Phorbol-13-Acetate DMBA-TPA

Two-stage skin carcinogenesis is induced by two topically applied chemicals. A mutagen 7,12-dimethylbenz[a]anthracene) causes mutations in the epidermal cells and a continuous application of general growth stimulator 12-O-tetradecanoyl phorbol-13-acetate accelerates skin papilloma formation.

使用二甲基苯甲酸酯和12-O-Tetradecanyl Phorbol-13-醋酸酯（DMBA-TPA）的化学诱导皮肤致癌模型

Cancer is one of the most devastating human diseases. Experimental cancer models are important to gain insight into the complex interplay of different ...

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, there is no effective long-term treatment for vitiligo. Therefore, it is imperative to develop a convenient and effective treatment for vitiligo. Regenerative medicine technology for direct reprogramming of skin cells into melanocytes seems to be a promising novel treatment of vitiligo. This involves the direct reprogramming of the patient's skin cells into functional melanocytes to help ameliorate the loss of melanocytes in patients with vitiligo. However, this method needs to be first tested on mice. Although direct reprogramming is widely used, there is no clear protocol for direct reprogramming into melanocytes. Moreover, the number of available transcription factors is overwhelming. 
Here, a concentrated lentivirus packaging system protocol is presented to produce transcription factors selected for reprogramming skin cells to melanocytes, including Sox10, Mitf, Pax3, Sox2, Sox9, and Snai2. Mouse embryonic fibroblasts (MEFs) were infected with the concentrated lentivirus for all these transcription factors for the direct reprogramming of the MEFs into induced melanocytes (iMels) in vitro. Furthermore, these transcription factors were screened, and the system was optimized for direct reprogramming to melanocytes. The expression of the characteristic markers of melanin in iMels at the gene or protein level was significantly increased. These results suggest that direct reprogramming of fibroblasts to melanocytes could be a successful new therapeutic strategy for vitiligo and confirm the mechanism of melanocyte development, which will provide the basis for further direct reprogramming of fibroblasts into melanocytes in vivo.

Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.

将小鼠成纤维细胞直接重编程为黑素细胞

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, ...

发育生物学

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2R&#947;null (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Humanized mouse models provide a more accurate representation of the human immune microenvironment. This manuscript describes the process in which these models are created through a renal graft of human thymus, injection of human CD34+ cells, and the targeted delivery of human cytokine transgenes to promote CD34+ cell proliferation and differentiation.

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation ...

免疫与感染

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology.

The aim of this protocol is to induce transient in vivo production of nonlethal levels of reactive oxygen species (ROS) in mouse skin, further promoting physiological responses in the tissue.

通过可切换原卟啉IX依赖性原位活性氧物种光生成刺激小鼠皮肤中的干细胞壁龛和组织再生

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient ...

生物学

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

Y-27632 丰富成人皮肤组织中人类黑色素细胞的产量

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Chemical-induced Two-stage Skin Carcinogenesis Model: An Experimental In Vivo Mouse Model of Skin Cancer

Source: V&#228;h&#228;tupa, M. et al. <a href="https://www.jove.com/v/60445"> Chemical-Induced Skin Carcinogenesis Model Using Dimethylbenz[a]Anthracene and 12-O-Tetradecanoyl Phorbol-13-Acetate (DMBA-TPA)</a>. J. Vis. Exp. (2019)
This video describes the procedure to induce two-stage skin carcinogenesis to study tumor initiation by topical application of chemicals. In the first step, a mutagen, DMBA, is applied onto the skin of a mouse model which initiates tumor formation by causing mutations in the stem cells, followed by the application of a growth stimulator, TPA, which accelerates skin papilloma formation.

化学诱导的两阶段皮肤致癌模型:一种皮肤癌的实验性体内小鼠模型

Source: V&#228;h&#228;tupa, M. et al.  Chemical-Induced Skin Carcinogenesis Model Using Dimethylbenz[a]Anthracene and 12-O-Tetradecanoyl ...

JoVE Encyclopedia of Experiments

Skin Cancer

Animal model studies

Minimum Erythematous Dose Assay: A Method for Measuring UV Sensitivity in Mouse Models

Source: Amaro-Ortiz, A. et al. <a href="https://www.jove.com/v/50670"> Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model</a>. J. Vis. Exp. (2013)
This video describes a method to measure UV sensitivity in a humanized mouse model&#8212;a mouse model that mimics human skin. We determine the minimal erythematous dose (MED) of UV radiation that can cause erythema or edema on the skin. The method can help to find out the efficacy of drugs used to reduce UV sensitivity.

最小红斑剂量测定:一种在小鼠模型中测量紫外线敏感性的方法

Source: Amaro-Ortiz, A. et al.  Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model. J. Vis. Exp. ...