作者要感谢迪尔德丽·尼尔森博士的有益的意见和批评性阅读的手稿。这项工作是由美国国立卫生研究院拨款DE019244，DE019197，和DE021841 ML，F32DE02098001圣雅各福群会，和C06 RR015464大学奥尔巴尼分校，纽约州立大学。

该协议包含四个主要的步骤，在图1中所描绘的。所有步骤都全面详细的描述。腺病毒载体建设和病毒纯化，应提前进行的活体摘取器官在基因转导的解剖上皮细胞的雏形。所有BSL-2标准的安全防范措施时，应遵循工作的腺病毒。 1。小鼠胚胎颌下腺（SMG）收获和显微切割<ol><li>安乐死定时的怀孕雌性小鼠（封闭群CD-1或ICR）使用CO 2昏迷状态，其次是颈椎错位和收获字符串的小鼠胚胎在胚胎13天（E13，3-5上皮芽）以下程序批准的机构IACUC委员会。阴道塞发现这一天被指定为E0。在E12阶段时，有一个单独的主芽和茎裂刚刚开始，唾液腺也有所收获，并培养启动。冲锋枪在此之前的阶段需要不同的培养条件下比在此间表示。 </li><li>转移胚胎字符串到无菌的10厘米组织文化的菜肴装有25毫升的DMEM /火腿的F12中（F12）无酚红（Life Technologies公司），辅以100ü/ ml青霉素，和100微克/毫升链霉素（笔，链球菌， Life Technologies公司）。 </li><li>用无菌手术刀（11片扇叶），镊子（＃5，精细的科学工具，11252-20），从囊中取出胚胎成一个独立的10厘米菜含有25毫升的DMEM/F12 +笔链球菌。 </li><li> SEVER的的胚胎头与下颚的正下方有角度的切割。 </li><li>下一个立体解剖显微镜与透射光底座（尼康SMZ645或同等学历）使用切割低于上颌骨，下颌骨的头低隔离。将顶部的一个额外的一块玻璃上，而不是直接在显微镜基础的玻璃成分的组织。 </li><li>收集下颌骨3毫升DMEM/F12 +笔链球菌在无菌的35 mm组织培养皿中。 </li><li>一个颚切片放置在解剖显微镜载物台上，使舌是切片的底部上，但指向12:00点钟位置。 </li><li>使用两个交叉钳在剪运动的一面，删除和丢弃的下1/3 路的下颌骨片。 </li><li>将片90°的右侧（右手），使用的钳子剪切一半切片，切下颌骨片的顶部（不切割的舌头）。 </li><li>剥离多余的组织，并删除它，露出下面的冲锋枪。会有一个舌根附近的每一侧上。 </li><li>用一个钳子按住组织的舌头，使用产钳的舌头挑逗SMG距离。重复的其他腺体。 </li><li>到一个新的无菌35mm组织分为3毫升的DMEM/F12 +笔-链球菌收藏报错显微切割的唾液腺培养皿中。注:较大的颌下腺（3-5上皮芽）与关连较小的舌下腺（1芽）是可以培养的完整跳过步骤4和执行步骤4.1，4.3，4.4，如前所述，2,8。 </li></ol> 2。 SMG上皮萌芽分离<ol><li>地点5（或更多）唾液腺腺体到玻璃底部的，50毫米直径微孔培养皿（MatTek公司，P50G-1.5-14-F）含有200微升的Hank氏平衡盐溶液（HBSS中缺乏钙+ +或镁+ +， Life Technologies公司），含有0.4％（体积/体积）分散酶（Life Technologies公司，目录号17105-041），并孵育15分钟，在37℃下</li><li>温育后，用200μl的无菌移液管尖，小心吸出分散酶溶液，而不会干扰腺体。立即加入200微升的DMEM/F12培养基含有5％（重量/体积）BSA（DMEM/F12-BSA用0.22μm的过滤器，预先灭菌的），以中和分离酶。 </li> &lt;李在解剖显微镜下，用钳子细提示（＃5 Dumostar，精细的科学工具，11295-20），轻轻分开松动的间充质细胞各地SMG上皮芽和管道，照顾离开上皮细胞的完整性。 </li><li>预湿的无菌200微升移液器吸头与DMEM/F12-BSA媒体，轻轻移液管的上皮细胞到一个新的50毫米直径的微孔菜含有200μl的DMEM/F12 +笔链球菌的雏形。使用高品质的200微升移液器吸头与平滑的边缘。 </li><li>在菜含有间质片，丢弃任何非间充质组织的舌下腺和颌下腺任何分离的芽。 </li></ol> 3。腺病毒感染的上皮雏形<ol><li> 200微升稀释在DMEM/F12 +笔链球菌在一个离心管中，使所得到的溶液的滴度为1×10 8 -10 9 PFU就能景点的腺病毒的病毒感染的媒体。 "不同的病毒所需的最佳的效价可能会有所不同。重要的是要使用氯化铯（CsCl）纯化的病毒制剂的最佳吸收效率。 </li><li>从盘子里取出媒体包含5个上皮细胞的雏形。快速加入200μl的感染媒介，在任何时候都保持潮湿的雏形。采取必要的预防措施，在处理病毒和处置受污染的枪头。 10％的漂白粉溶液消毒任何被病毒污染的表面。 </li><li>将上皮细胞的雏形远从彼此用钳子夹，以防止它们粘在一起，使他们能够解决的盘底。在病毒性媒体，在室温下的1小时的孵育雏形。在培育，分离的雏形，如果他们靠拢在一起。过度摇摆或运动将使腺体聚集在一起。 </li><li>培养结束后，温和地去除病毒的媒体和丢弃适当，小心不要破坏了Rudiments。加入200μlDMEM/F12 +笔链球菌。重复此清洗至少两次，并更换200μl的DMEM/F12 +笔链球菌媒体。这些洗是关键，以消除任何病毒，不连接的上皮和间质，以防止感染。 </li><li>孵育上皮雏形与200毫升的DMEM/F12中含有2％（体积/体积）的基质胶（BD Biosciences公司，356231）20分钟。我们发现，这在Matrigel的潜伏期短，最大限度地减少了现有裂在最初的文化回归，但这个步骤缺一不可，如果不希望暴露在Matrigel的。 </li></ol> （4） 体外培养重组冲锋枪<ol><li>放置每个雏形，一次一个地，使用预湿的200μl的移液管尖，在顶部的切口（小切口）13毫米，0.1微米核孔履带蚀刻膜过滤器（Whatman）超过200μl的培养基（漂浮入门/过滤器）在玻璃底的微孔板。文化传媒是:含有50微克/毫升转铁蛋白和150微克/毫升的抗坏血酸，如前所述2,8的DMEM/F12 +笔-链球菌。转铁蛋白是众所周知的刺激的生存和分支的唾液腺等器官外植体的形态发生9。添加抗坏血酸已证明刺激SMG分支。10是已知的作为一个辅助因子在许多羟化反应的代谢途径（如脯氨酸羟化胶原纤维形成11期间），也刺激其他器官的外植体中的纤连蛋白和层粘连蛋白的生产12。 </li><li>后移除尽可能多的媒体之上的过滤器尽可能增加每个上皮雏形。太多媒体的过滤器上，可能会导致过滤器下沉，而较少的媒体位置的雏形和间充质更容易。可以去除多余的介质，使用了一套吸头或用钳子夹住媒体之间的技巧。 </li><li>地点间充质件的来回米3至4个腺体的顶部和/或围绕每个上皮雏形。删除任何额外的介质，周围的腺体，间质，以避免运动距离的雏形。间充质来自更腺体的是没有好处的，但不太可能会损害上皮雏形增长。 </li><li>孵育重组冲锋枪可增湿培养箱中（95％空气/ 5％CO 2）在37℃下为48 -72小时，根据需要。 </li><li>收购明场和/或荧光图像的活腺体0小时（如果需要），重组后2小时，每24小时后倒置，直立，或立体光学显微镜配备了一台数码相机使用了4倍或5倍放大倍率的目标是在一个单一的框架来看，捕捉整个重组腺。唾液腺的图像显示使用明场设置或适当的相环的光路（上完全自动化的显微镜，这是不可能的）部分进入最佳的对比度。标准相差不吨必要的，因为腺体很厚，产生的对比。 </li><li>在期望的时间点可以是固定的20分钟制成的2％多聚甲醛（PFA）的1X PBS中含有5％（重量/体积）蔗糖（从而更好地保持的保持的细胞在细胞内部结构与固定液，重组冲锋枪等渗状态）。不同于4％PFA，2％PFA将维持一个显着量的GFP信号，如果固定剂固定后完全除去。腺体，可存储长达1周1X PBS在黑暗中，在4°C，直到整个安装进行免疫细胞化学和激光共聚焦成像，如先前报道3,6,13-15。理想的情况下，免疫细胞化学和成像应尽快进行固定后，以获得最佳的图像质量。腺体也可以在RIPA缓冲液中溶解，然后通过Western印迹法分析进行SDS-PAGE。 </li></ol>

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没有利益冲突的声明。

上皮-间质体外重组技术首次公布于1981年16颌下唾液腺。在这个协议中，我们扩大后，原来的方法，使用腺病毒感染的操作范围内的重组腺上皮细胞基因的表达。我们表明，与腺病毒感染的上皮细胞的百分比，而被感染的细胞的百分比取决于病毒启动子的性质，病毒滴度，和病毒纯度。我们已经发现，有必要使用CsCl梯度纯化的高效上皮细胞转导，其中不在这里描述的纯化的病毒。既...

<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 </td></tr><tr><td>无酚红DMEM /火腿的F12中</td><td> Life Technologies公司</td><td> 21041-025 </td><td></td></tr><tr><td>青霉素和链霉素</td><td> Life Technologies公司</td><td> 15070-163 </td><td> 10倍的股票</td></tr><tr><td>中性蛋白酶</td><td> Life Technologies公司</td><td> 17105-041 </td><td>冻结一次性使用等份-20C </td></tr><tr><td> BSA </td><td>西格玛</td><td> A2934-100G </td><td>分数V，低内毒素</td></tr><tr><td>腺-X-GFP的</td><td> BD Biosciences公司</td><td> 8138-1 </td><td>应该是高滴度的（1×10 10 pfu /毫升）。 CsCl纯化病毒是更有效地比在该测定中的柱纯化病毒。 </td></tr><tr><td> 16％的多聚甲醛</td><td&gt;电子显微学</td><td> 15710 </td><td>在PBS中稀释至2％，用5％蔗糖（w / v的） </td></tr><tr><td> 1X磷酸盐缓冲盐水（PBS） </td><td> Life Technologies公司</td><td> 70011-044 </td><td>准备从10倍的股票</td></tr><tr><td> Hank氏平衡盐溶液</td><td> Life Technologies公司</td><td> 14175095 </td><td>没有钙，镁，没有酚红</td></tr><tr><td>转</td><td>西格玛</td><td> T8158 </td><td> 25毫克/毫升股票在DMEM/F12媒体解决方案。冻结一次性使用的分装-20C </td></tr><tr><td> L-抗坏血酸（维生素C） </td><td>西格玛</td><td> A4403 </td><td> 75毫克/毫升原液在DMEM/F12 media.Freeze的单次使用的等份-20C </td></tr><tr><td></td><td></td><td></td><td> 表1列出的，为SMG重组协议所需的试剂。 </td></tr><tr><td></td><td></td><td></TD&gt; <td></td></tr><tr><td> 10厘米无菌塑料菜</td><td>康宁</td><td> 430167 </td><td>非组织培养处理的板，也可以被使用。 </td></tr><tr><td>立体解剖显微镜透射光底座</td><td>尼康</td><td> SMZ645 </td><td>可以使用具有任何立体解剖显微镜的透射光基础。 </td></tr><tr><td> 35 mm组织培养皿</td><td>鹰</td><td> 353001 </td><td>非组织培养处理的板，也可以被使用。 </td></tr><tr><td> 50毫米直径的微孔菜</td><td> MatTek公司</td><td> P50-G-1.5-14F </td><td></td></tr><tr><td>核孔径迹蚀刻膜过滤器</td><td> Whatman公司</td><td> 110405 </td><td> 13毫米直径，0.1毫米孔径</td></tr><tr><td>宽视场荧光显微镜</td><td>卡尔·蔡司，美国</td><td> Axio上观察Z1 </td><td>任何荧光显微镜（正直，我nverted或立体解剖显微镜）可以与一个附加的数码相机，用于监视在低放大倍率的GFP表达。 </td></tr><tr><td>共聚焦显微镜</td><td>徕卡显微系统</td><td> TCS SP5 </td><td>激光共聚焦显微镜是要看到详细的细胞结构。在任何可以使用共聚焦显微镜。 </td></tr><tr><td>定时怀孕的雌性小鼠，CD-1菌株或ICR </td><td>查尔斯河实验室</td><td></td><td>收获胚胎第13天（天插头发现指定为第0天）。 </td></tr><tr><td>手术刀＃11 </td><td>精细科学工具</td><td> 10011-00 </td><td></td></tr><tr><td>手术刀手柄＃3 </td><td>精细科学工具</td><td> 10003-12 </td><td></td></tr><tr><td>杜蒙＃5镊子抗氧化合金，0.05mm的X0.02毫米</td><td>精细科学工具</td><td> 11252-20 </td><td>从胚胎收获腺体的理想选择</td></tr><tr><td&gt;杜蒙第5的镊子dumostar合金，0.05mm的X0.01毫米</td><td>精细科学工具</td><td> 11295-20 </td><td>精细的技巧都需要消除从上皮细胞的间充质。也可以使用钨针。 </td></tr><tr><td></td><td></td><td></td><td> 表2。使用的设备，在SMG重组协议。 </td></tr></tbody></table>

genetic modification and recombination of salivary gland organ cultures

分支形态发生发展过程中的许多器官和胚胎小鼠颌下腺（SMG），是一个经典模型的研究分支形态。在发展中国家SMG，这个过程涉及迭代步骤的上皮芽管形成，最终上升到一个复杂的腺泡和导管，分支网络的服务产生和修改/运输唾液，分别进入口腔1 - 3。上皮相关的基底膜和间充质室，包括间充质细胞，生长因子和细胞外基质，由这些细胞产生的方面，分支机构是至关重要的，但如何协调细胞和分子事件仍然知之甚少4 。这项研究的分子机制驾驶上皮形态的进步，我们的发展机制的理解和洞察可能雷格纳积极行动的医学方法。这些研究的唾液腺上皮细胞进行遗传操作的有效方法由于缺乏而受到阻碍。目前，腺病毒转导针对成年腺体的上皮细胞在体内 5表示的最有效方法。然而，在胚胎外植体，致密的间充质细胞和上皮细胞周围的基底膜阻碍病毒获得的上皮细胞。如果间质被除去，被转染的上皮细胞可以使用腺病毒，和上皮雏形可以恢复分支形态存在的Matrigel或层粘连蛋白-111 6,7。间充质-上皮雏形的增长还需要额外的补充可溶性生长因子，并不能完全概括分支形态，因为它发生在完整的腺体8。在这里，我们描述了一种技术，这有利于腺病毒转导的上皮细胞和转染的电子文化pithelium相关的间充质。胚胎冲锋枪，去除间质，用含GFP-腺病毒感染的上皮细胞显微切割之后，我们显示，上皮细胞与未感染的间充质细胞自发重组，扼要完整SMG腺体结构和分支形态。使用标准的荧光显微镜方法，可以很容易地监控转基因上皮细胞人口，如果荧光标记的腺病毒结构。这里描述的组织重组方法是目前最有效的和可访问的方法，与野生型或突变型的矢量在一个复杂的三维组织结构，不需要产生转基因动物的上皮细胞的转染。

流动的主要的实验步骤在图1中概述。在图2中所示的一个例子的一个完整的SMG，一个孤立的上皮雏形，和其相应的间充质细胞。固定和成像的明视场图像重组冲锋枪，继续进行培养体外时的指定的时间，在图3中所示的分支形态，生长48小时表达上皮的GFP的图4中所示的重组质粒腺体的重组冲锋枪的共聚焦图像随后通过免疫细胞化学的72小时培...

Watch this Scientific Journal Video about Genetic Modification and Recombination of Salivary Gland Organ Cultures at JoVE.com

Genetic Modification and Recombination of Salivary Gland Organ Cultures

一个基因操纵技术上皮细胞内全<em&gt;体外</em培养的胚胎小鼠颌下腺（冲锋枪）病毒转基因。这种方法利用的先天能力，SMG上皮细胞和间充质，自发重组后的分离和感染的上皮与腺病毒载体的雏形。

基因改造和重组唾液的腺体器官培养

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

genetic-modification-recombination-salivary-gland-organ

Research

JoVE Journal

Biology

12.8K 次观看.  University at Albany, SUNY. 一个基因操纵技术上皮细胞内全体外

视频: Genetic Modification and Recombination of Salivary Gland Organ Cultures

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol demonstrates dissection techniques for removal of the midgut and salivary glands from Aedes aegypti mosquitoes. 



The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

从AE肠和唾液腺的解剖。埃及伊蚊蚊

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

科研

生物学

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their head and neck regions for cancer treatment. Both groups of patients experience symptoms such as xerostomia (dry mouth), dysphagia 
(impaired chewing and swallowing), severe dental caries, altered taste, oro-pharyngeal infections (candidiasis), mucositis, pain and discomfort.
One innovative approach of regenerative medicine for the treatment of salivary gland hypo-function is speculated in RS Redman, E Mezey 
et al. 2009: stem cells can be directly deposited by cannulation into the gland as a potent method in reviving the functions of the impaired organ. Presumably, 
the migrated foreign stem cells will differentiate into glandular cells to function as part of the host salivary gland. Also, this cannulation technique is an 
expedient and effective delivery method for clinical gene transfer application.
Here we illustrate the steps involved in performing the cannulation procedure on the mouse submandibular salivary gland via the Wharton's duct 
(Fig 1). C3H mice (Charles River, Montreal, QC, Canada) are used for this experiment, which have been kept under clean conventional conditions at 
the McGill University animal resource center. All experiments have been approved by the University Animal Care Committee and were in accordance with the 
guidelines of the Canadian Council on Animal Care.
For this experiment, a trypan blue solution is infused into the gland through the opening of the Wharton's duct using a 
insulin syringe with a 29-gauge needle encased inside a polyethylene tube. Subsequently, the mouse is dissected to show that the infusions migrated 
into the gland successfully.

A protocol for the cannulation of the mouse submandibular salivary gland via the Wharton's duct is described. For this experiment, the trypan blue solution is used as a dyer to demonstrate how this technique effectively delivers infusions into the targeted gland, and to suggest the reliability of this new approach as a potential clinical drug/cell therapy for the regeneration of salivary glands.

沃顿商学院的导管插管通过小鼠颌下腺唾液腺

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their ...

医学

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

鼠标涎腺干细胞的分离

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

从人类唾液腺中分离唾液表皮细胞,作为脂质层或单层体进行体外生长

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

免疫与感染

Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In these cases, questions remain regarding disease pathogenesis and effective interventions. An optimized technique that allows functional assessment of the salivary glands is invaluable for investigating exocrine gland biology, dysfunction, and therapeutics. Here, we present a step by step approach to performing pilocarpine stimulated saliva secretion, including tracheostomy and the dissection of the three major murine salivary glands. We also detail the appropriate murine head and neck anatomy accessed during these techniques. This approach is scalable, allowing for multiple mice to be processed simultaneously, thus improving the efficiency of the work flow. We aim to improve the reproducibility of these methods, each of which has further applications within the field. In addition to saliva collection, we discuss metrics for quantifying and normalizing functional capacity of these tissues. Representative data are included from submandibular glands with depressed salivary gland function 2 weeks following fractionated radiation (4 doses of 6.85 Gy).

We present a detailed approach to performing saliva collection, including murine tracheostomy and the isolation of three major salivary glands.

小鼠涎腺照射后通过匹属刺激进行唾液功能评估

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In ...

Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery

Salivary glands are an attractive tissue target for gene therapy with promising results already leading to human trials. They are inherently capable of secreting proteins into the bloodstream and are easily accessible, making them potentially superior tissue sites for replacement hormone production or vaccination by gene transfer. Suggested methods for gene delivery include transcutaneous injection and retrograde infusion through salivary ducts. We demonstrate how to perform Retrograde Salivary Gland Infusion (RSGI) in non-human primates. We describe the important anatomic landmarks including identification of the parotid papilla, an atraumatic method of cannulating and sealing Stensen&#39;s Duct utilizing basic dental tools, polyethylene tubing, and cyanoacrylate, and the appropriate rate of infusion. While this is the least traumatic method of delivery, the method is still limited by the volume able to be delivered (&#60;0.5 mL) and the potential for trauma to the duct and gland. We demonstrate using fluoroscopy that an infusate can be fully delivered into the gland, and further demonstrate by immunohistochemistry the transduction of a typical vector and expression of the delivered gene.

Salivary glands have been proposed as a tissue target site for gene therapy, especially in the area of vaccination by gene transfer. We demonstrate gene delivery in a non-human primate model utilizing retrograde parotid infusion.

逆行帕罗蒂德格兰德输液通过斯滕森的管道在非人类灵长类病原体矢量基因交付

Salivary glands are an attractive tissue target for gene therapy with promising results already leading to human trials. They are inherently capable of ...

Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.

Using three-dimensional organotypic cultures to visualize morphology and functional markers of salivary glands may provide novel insights into the mechanisms of tissue damage following radiation. Described here is a protocol to section, culture, irradiate, stain, and image 50&#8211;90 &#956;m thick salivary gland sections prior to and following exposure to ionizing radiation.

下颌骨和腮腺表型模型对组织型培养的放射治疗是机体活力特征的关键

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with ...

小鼠下颌下腺再生的实时实时成像

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.
The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

作者聚焦:研究损伤后唾液腺再生中巨噬细胞-上皮细胞的相互作用 

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

通过离体活细胞成像询问唾液腺中的细胞间相互作用

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal ...

基因改造和重组唾液的腺体器官培养

摘要

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此视频中的章节

从AE肠和唾液腺的解剖。埃及伊蚊蚊

沃顿商学院的导管插管通过小鼠颌下腺唾液腺

鼠标涎腺干细胞的分离

从人类唾液腺中分离唾液表皮细胞,作为脂质层或单层体进行体外生长

小鼠涎腺照射后通过匹属刺激进行唾液功能评估

逆行帕罗蒂德格兰德输液通过斯滕森的管道在非人类灵长类病原体矢量基因交付

下颌骨和腮腺表型模型对组织型培养的放射治疗是机体活力特征的关键

通过离体活细胞成像询问唾液腺中的细胞间相互作用

通过离体活细胞成像询问唾液腺中的细胞间相互作用

通过离体活细胞成像询问唾液腺中的细胞间相互作用