Name: surgical injury to the mouse pancreas through ligation of the pancreatic duct as a model for endocrine and exocrine reprogramming and proliferation
Uploaded: 2015-08-07T13:00:00.000+00:00
Duration: 7 min 44 s
Description: Watch this Scientific Journal Video about Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation at JoVE.co

The authors acknowledge all colleagues who made this work possible; Ann Demarr&#233;, Veerle Laurysens, Jan De Jonge and Erik Quartier for technical assistance. Financial support was from the VUB Research Council, the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT), the Beta Cell Biology Consortium (BCBC), the Fund for Scientific Research Flanders (FWO), Diabetes Onderzoek Nederland (DON) and Interuniversity Attraction Pole (IAP).

所有操作遵循保护脊椎动物用于试验和其他科学目的发行的欧洲公约的准则（ETS 123和2010/63 / EU）。 1.准备工作区<ol><li>提供专用的准备区，给外科手术区域和回收区。 </li><li>进行整个手术过程中的层流柜，以尽量减少环境污染物。 </li><li>组装用品所需的制备，手术和恢复区（如在材料和方法中列出），采用适当的无菌技术。 </li><li>确保手术工具在手术前高压灭菌。 </li><li>提供一个再循环水加热垫在38℃的温度稳定在手术过程中的温度。覆盖加热垫用无菌防水垫。 </li><li>提供一种手术显微镜用至少6.3X的倍率。 </li><li>使用仪器免缝lizer，如热珠灭菌，消毒器械在外科手术过程之间。 </li><li>准备恢复区组成的大笼子中，由平板纸床上用品内衬</li></ol> 2.准备动物外科手术<ol><li>对于PDL和假手术，使用8个周龄雄性小鼠，圈养在标准笼子里，维持12小时光照/ 12小时黑暗循环，并饲喂标准的啮齿动物饮食随意 。 注:在这里，我们用BALB / cJrJ小鼠，但我们也成功地使用其他的菌株和各种转基因品系。 </li><li>丁丙诺啡使用作为超前镇痛（0.05 - 0.1毫克/千克）30分钟前手术。 </li><li> 10mg / kg的甲苯噻嗪 - 通过腹膜内注射100mg / kg的氯胺酮和5麻醉小鼠。 </li><li>通过观察随意运动和肌肉松弛逐渐丧失正确评估麻醉。测试反射由脚趾捏的损失。 </li><li>应用眼药膏上一页眼睛干涩耳鼻喉科，而在麻醉下。 </li></ol> 3.手术场地准备<ol><li>消毒胸部和腹部用消毒洗必泰溶液。 </li><li>剃为2.5cm×1.5cm的腹部的区域。 </li><li>用消毒纱布浸泡洗必泰溶液，再用酒精溶液和洗必泰溶液的最终应用领域剃。 </li><li>定位动物在手术区，使得所制备的手术部位向上面对外科医生。 </li><li>使用防水外科被单有开口留下露出而覆盖身体的其他部分来创建一个无菌工作场的消毒腹部区域悬垂鼠标。监测之前对麻醉深度的方法的鼠标。 </li></ol> 4.胰管结扎<ol><li>剖腹手术<ol><li>使上部中线切口，在皮肤用无菌从剑突延伸到肚脐手术刀片。分离底层白线，并使用无菌剪刀以暴露上腹部象限腹膜。 </li><li>防止干燥出内脏定期喷洒消毒用0.9％氯化钠。 </li><li>使用无菌镊子或拭子，缩回胃优，露出脾和胰腺的脾叶（尾区）。 </li><li>轻轻缩回空肠上段的十二指肠和部分向右上腹部象限以暴露覆盖由内脏腹膜胰腺的头部，颈部和主体区域。 </li></ol></li><li>结扎<ol><li>定位在胰腺的颈部区域中的胰主导管的解剖位置。 </li><li>切开脏腹膜和结肠韧带，准许访问腹膜后，露出胰腺的主体和尾部区域。为了暴露颈部区域，执行科赫尔机动。提起十二指肠和头部关闭腹膜后胰腺从下腔静脉和主动脉之下他们提升。 </li><li>小心地将颈部下方的铲。结扎胰管用6-0 Prolene线螺纹在门静脉分隔胃十二指肠和脾瓣的左侧。 </li><li>执行在靠近第二结扎到第一结扎，以确保瓣适当分开。 </li><li>结扎非常小心，以便不损坏下层的血管，即上级胰十二指肠动脉，胰十二指肠下动脉和脾动脉的胰一部分。 </li><li>将器官放回腹腔。 </li><li>关闭使用4-0聚乙二醇丝状螺纹切口以连续缝合图案为肌肉/腹膜层和在对皮肤的不连续的缝合图案。 </li></ol></li></ol> 5.假手术<ol><li>执行所有的步骤去4.1.4通过划线步骤1。而胰腺暴露，不执行胰管结扎。 </li><li>在步骤4.1.4结束时，将内脏放回腹腔。 </li><li>关闭切口在4.2.7中描述。 </li></ol> 6，后期运营维护和监控<ol><li>手术过程完成后，将鼠标在回收区。这包括一个笼子放置在一个加热垫，并镶有平纸张的床上用品，以保持正常的体温。 </li><li>提供营养支持，以避免手术后的低血糖蘸通过食物放置在笼底。为了这个目的，用浸在水中的标准啮齿动物饮食粒料直到它们变软。 </li><li>由食品润湿液提供支持，并提供水随意 。 </li><li>使用丁丙诺啡作为镇痛（0.05 - 0.1毫克/公斤），每天两次2天手术后。在整个实验，跟进，observE中的小鼠发生感染的可能迹象，包括分泌液体或脓液从伤口，或用于修饰行为和活动水平的特点是减少身体每况愈下，食欲降低体重和损失。 </li></ol> 7.成功的评价PDL和PDL尾的收获和深尾胰腺<ol><li>安乐死的小鼠通过颈脱位。 </li><li>再剃腹部，避免皮草结转。 </li><li>打开腹部皮肤和肌肉层和除去大面积皮肤和肌肉得到良好的访问胰腺。 </li><li>使用无菌镊子或拭子，胃被优缩回，露出脾和胰腺的脾叶（尾区）。 </li><li>轻轻拉出空肠上段的十二指肠及部分露出胰腺的胃十二指肠叶（头部区域）。 注意:将PDL胰腺的连接部分现在已减小尺寸，并已成为几乎半透明使胰岛白色小圆点可见。胰腺的头部是不透明的粉红色和不同外分泌lobuli可以观察到。 </li><li>使用无菌剪刀，从脾脏中分离的结扎尾巴部分的胰腺，通过沿脾切割。切松结缔组织尾部区域胰腺连接到内部器官。 </li><li>切PDL尾部区域正好在结扎前，但不包括的绑带与所述组织立即从收获的组织邻接它。 </li><li>为了隔离假尾巴组织，按照步骤7.1至7.5。用无菌剪刀沿脾切分开尾部区域的假胰腺脾脏。切成胰腺的颈部区域和切割松开结缔组织尾部胰腺连接到内脏隔离尾部区域 注:假胰腺的两个尾部和头部区域是不透明的粉红色，分别隔离两个部分，切在颈部区域中的胰腺。 </li></ol>

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The authors have nothing to disclose.

In the present study, we describe in detail the methodology behind PDL, a mouse injury model to study beta cell neogenesis and proliferation and transdifferentiation of pancreatic non-beta cells. Ambiguity in data on PDL among labs stimulates the need for a standardized protocol for PDL surgery.
Critical steps in the PDL protocol include the selection of healthy, young mice for surgery. Preferably male mice should be used since unpublished data from our lab suggest that estrogen receptor signaling affects the outcome of PDL. PDL surgery induces recruitment and expression of many factors to/in the injured pancreas and dissection of this cocktail may allow the identification of factors that are necessary and sufficient to cause beta cell neogenesis and proliferation to bypass the need for surgery. However, it is important to realize that the cytokines and factors involved in this model are not yet completely characterized. Therefore, it is important to perform PDL surgery under optimal conditions since severe infection or disease of the animal can alter the occurrence of these factors and thereby affect the outcome of PDL. We advise to monitor the animals closely after PDL surgery by assessing weight, glycemia, appetite, and activity level.
Proper ligation of the pancreatic duct is the most crucial step in the protocol. The ligation needs to be secured with multiple loops to prevent incomplete ligation that may reduce the severity of the injury. However, the ligation should only affect the pancreatic duct, avoiding the underlying superior and inferior pancreaticoduodenal artery. Accidental ligation of the arteries can lead to accessory damage and to hypoxia of the pancreas with possible subsequent necrosis. 
A sub-optimal PDL can occur due to improper ligation of the duct. This leads to a remnant of acinar tissue, as evidenced at day 7 and 14 after PDL surgery. We advise not to include sub-optimal PDL samples into the analysis, since this can lead to variation in analyzed parameters such as beta cell proliferation and Ngn3 expression. Notably, the level of Ngn3 expression correlates with the level of beta cell neogenesis.15 [Hansen M.T., et al., in preparation]. A successful PDL can be recognized by the visible reduction in size and transparency of the tissue compared to a non-ligated pancreas, phenomena which are clearly visible at 7 and 14 days post-surgery. Histological analysis should reveal fibrotic tissue, increased number of ductal structures, increased ductal and endocrine proliferation and recruitment of immune cells. RT-qPCR should reveal an expression of Ngn3 in PDL tail of at least 20% of the expression level in duodenum.
When analyzing proliferation in a non-automated manner by individual inspection of cells, the duct and endocrine cell proliferation in PDL leads to very reproducible results. The induction of Ngn3 expression, however, varies between experiments, indicating that this process may be more sensitive to physiological and environmental factors. Since PDL induces recruitment and/or activation of many cells and factors, identification of the factor(s) responsible for a certain process in PDL is challenging. 
As a model in diabetes research, PDL does not require removal of pancreatic tissue or of beta cell reduction, as compared to pancreatectomy, alloxan or streptozotocin injection. Therefore, animals usually maintain good glycemic control after PDL surgery. It can be expected that beta cell proliferation and activation of an Ngn3-expressing progenitor is stimulated by locally produced signals originating from the inflammatory responses and from acinar cell death. Therefore, PDL is an injury model that can serve as an analytical tool to study signaling pathways involved in beta cell formation. Moreover, the experimental model of PDL may be useful beyond diabetes research: while being artificial, the inflammatory nature of PDL mimics human pathological conditions and has therefore been used as a model to study pancreatitis31, formation of adenocarcinoma32 and neoplasia.33

糖尿病的患病率增加，影响超过3亿人口的世界级1,2提振了寻找新产生胰岛素的β细胞的来源，无论是在体外和体内，以补充不足的β细胞质量。3识别关键机制和因素调节β细胞增殖和β细胞新生， 即 β细胞从非β细胞或祖细胞的分化，可以用于再生治疗的糖尿病的发展提供了新的目标。 在显影啮齿动物胰腺，所有的内分泌细胞类型，从内分泌祖细胞的流动人口区分，表达转录因子Neurogenin3（Ngn3的）。4,5在成年啮齿动物胰腺，在正常生理条件下，β细胞量是保持在一个最佳的数量，以满足代谢需求。改变β细胞的大小，凋亡和复制构成为β细胞扩张和关上。6-8的主要机制虽然β细胞的潜力，在正常生理条件下增殖是整个人口均匀的，9其增殖率低和再复制是通过限制由于内分泌祖细胞迄今尚未在正常成人胰腺确定一个动态静止期间或不应期6,7，按年龄和葡萄糖代谢的影响。10，新生被认为不利于正常成人的β细胞的生长。8 因此，在成人胰腺即膨胀并能产生新的β细胞将提供一种新的，β细胞可能无限来源的兼性内分泌祖细胞的鉴定。 局部管结扎（PDL）是已经描述以诱导β细胞neogenesi动物损伤模型S在成人胰腺11,12在此模型中，主胰管引流胰尾被手术结扎。外分泌引流阻塞造成重大诱导组织重塑，伴有炎症和腺泡萎缩远端结扎12-14在这种炎症环境中，重新表达的内分泌祖标记Ngn3的是诱导β细胞的体积会增加两倍。此加倍的β细胞量的结果从新的β细胞的产生由一个Ngn3的表达胚胎型内分泌祖细胞，并从预先存在的和新形成的β细胞，它们很容易重新复制没有"静止时期"的增殖。 11,15 β细胞新生和复制在损伤模型，如胰腺6,7,16-19和β细胞20的选择性消融已被广泛地描述。但是，在采取这些再生结果E型是由造成损伤的程度的影响，与降低的初始β细胞量21相关联。 PDL是其中最初的β细胞的质量不会受到影响，β细胞新生和增殖被鲁棒地激活一个外科模型。事实上，在小鼠接受PDL的胰腺，Ngn3的表达细胞被识别为靠近管道的上皮衬里。这些细胞可以从Ngn3的-GFP转基因小鼠的结扎胰腺使用荧光激活细胞分选（FACS）分离，并能够以下植入入和离体 E12.5 Ngn3的胰腺的培养分化朝官能β细胞- / - 。小鼠11同样，在Ngn3的CreERT; R26 YFP小鼠中细胞激活基因Ngn3的他莫昔芬注射，标签阳性Ngn3的细胞衍生的β细胞被PDL后发现后都将被永久标记15。此外，新成立的β细胞预稀释-exist荷兰国际集团的β细胞，并优先在找到小胰岛在其中β细胞显示出高增殖潜力。15 Ngn3的是PDL后β细胞扩张很重要，因为使用目标特定的短发夹RNA显著减少β细胞大规模和β细胞的增殖后下降Ngn3的表达PDL 11值得注意的是，Ngn3的细胞来源的β细胞的PDL后的级分和β细胞量的关键取决于Ngn3的感应15的水平。这是根据该观察结果Ngn3的表达的高水平是用于从多能胰祖细胞胰腺发育过程中的内分泌承诺的一个关键步骤22此外，Ngn3的表达细胞由白喉毒素给药于Ngn3的CreERT选择性烧蚀; R26 IDTR小鼠结果在降低的胰岛素含量和降低的β细胞增殖，特别是在小的胰岛15 <p类="jove_content"&gt;虽然在PDL后管细胞Ngn3的表达诱导已经证实了许多11,15,16,23,24，在胰岛细胞24,25和差异Ngn3的表达PDL的结果挑战我们的初步意见增加β细胞量26,27，外观Ngn3的表达的管道源性内分泌祖细胞24,26,28,29，并增加β细胞的增殖27 PDL之后。30 这些相互矛盾的结果可能，至少部分地，被最重要的归因于各种因素，包括变化的小鼠，手术后的生理和环境条件，并进行分析，体重，性别和年龄的手术后的时间点的组合，差异手术技术。30在我们的手中，β细胞增殖，胰岛素含量，β细胞体积和小岛的数量PDL后持续增加。还NGN3 mRNA的持续增加，但在PDL胰腺尾部，为此我们没有直接的解释之间的Ngn3的表达差异较大。我们假设，Ngn3的 mRNA的水平可能与来自非β细胞15的β细胞新生的程度相关，但这需要进一步属实。虽然它不会删除所有的实验变化，标准化的方法进行手术PDL允许在结果更好的一致性，并在研究β细胞的增殖和新生开辟了新的途径。

<table><tbody><tr><td>Supplies for preparation area&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Hibiscrub&amp;nbsp;</td><td>Regent Medical</td><td>5601IE5F11</td><td>Chlorhexidin diglucon</td></tr><tr><td>Ketamin</td><td>Ceva</td><td>BE-V202526</td><td>anesthesia</td></tr><tr><td>Rompun(2%) (Xylazin)</td><td>Bayer</td><td>BE-V041815</td><td>anesthesia</td></tr><tr><td>Duratears</td><td>Alcon</td><td>34335-8</td><td>ointment for eyes</td></tr><tr><td>Razor</td><td></td><td></td><td></td></tr><tr><td>Supplies for surgical area&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Leica Operating microscope</td><td>Leica</td><td>10446320</td><td></td></tr><tr><td>Hot bead sterilizer</td><td>Fine Science Tools (FST)</td><td>18000-45</td><td></td></tr><tr><td>autoclaved surgical instruments</td><td>Fine Science Tools (FST)</td><td></td><td></td></tr><tr><td>Recirclulating water heating pad</td><td>Gaymar Industries, Inc.</td><td>TP702</td><td></td></tr><tr><td>Adhesive OP-towel</td><td>BARRIER</td><td>706500-07</td><td></td></tr><tr><td>OP-tape</td><td>BARRIER</td><td>381035-00</td><td></td></tr><tr><td>Stella 3/5 Compresse de gaze (Sterile)</td><td>Lohmann&amp;amp;Rauscher International</td><td>35968</td><td></td></tr><tr><td>Mini Plasco 0.9% NaCl solution</td><td>B.BRAUN</td><td>3521680</td><td></td></tr><tr><td>6-0 prolene suture</td><td>Ethicon</td><td>8706H</td><td></td></tr><tr><td>4-0 polyglycol suture</td><td>Ethicon</td><td>SL-607</td><td></td></tr><tr><td>Supplies for recovery area</td><td></td><td></td><td></td></tr><tr><td>Paper bedding (paper towel)</td><td></td><td></td><td></td></tr><tr><td>Vetergesic</td><td>ecuPhar</td><td>BE-V342955</td><td></td></tr><tr><td>Materials for analysis</td><td></td><td></td><td></td></tr><tr><td>Taqman &lt;i&gt;Ngn3&lt;/i&gt; primer</td><td>Integrated DNA technologies</td><td>Mm.PT.56a.33574796.gs</td><td></td></tr><tr><td>Taqman CycloA primer</td><td>Integrated DNA technologies</td><td>Mm.PT.39a.2.gs</td><td></td></tr><tr><td>anti-Rabbit Cleaved Caspase 3 antibody (D175)</td><td>Cell Signaling</td><td>5A1E</td><td></td></tr><tr><td>anti-mouse/rat Ki-67 antibody</td><td>eBioscience</td><td>14-5698-82</td><td></td></tr><tr><td>monoclonal anti-actin alpha-Smooth muscle-Cy3 (mouse)</td><td>Sigma</td><td>085K4889</td><td></td></tr><tr><td>anti-rat Cytokeratin 19&amp;nbsp;</td><td>DSHB</td><td></td><td></td></tr><tr><td>monoclonal Mouse anti-BrdU</td><td>Dako</td><td>M0744</td><td></td></tr><tr><td>Hoechst</td><td>Sigma</td><td>33342</td><td></td></tr></tbody></table>

surgical injury to the mouse pancreas through ligation of the pancreatic duct as a model for endocrine and exocrine reprogramming and proliferation

扩张胰腺β细胞在体内或体外 ，或产生β细胞通过分化从胚胎或成体干细胞，可以提供β细胞的新扩展的来源，以减轻在人类胰岛移植的治疗糖尿病的供体短缺。虽然最近已经取得进展实现这一目标，调节β细胞扩增和分化的干/祖细胞的机制仍有待特点。在这里，我们描述了一个协议，用于在成年小鼠胰腺可作为研究组织重构和β细胞的增殖和分化的机制的工具功能的损伤模型。局部管结扎（PDL）是啮齿动物胰腺涉及造成阻塞的外分泌产物引流出来的尾部区域胰腺的主胰管手术结扎的实验诱导的损伤。在造成损伤诱导腺泡萎缩，免疫细胞infilt口粮和严重组织重塑。我们以前曾报道神经元素（NGN）3的活化表达内源祖细胞样细胞和PDL后增加β细胞增殖。因此，PDL提供了基础，研究涉及β细胞动力学信号，并在成人胰腺内分泌祖细胞的性质。因为，它仍然在很大程度上仍然不清楚，哪些因素和途径促进β细胞新生和增殖的客运专线，标准化的协议将PDL允许跨实验室比较。

PDL诱导腺泡萎缩和炎症，但不影响体重和血糖 在第8周龄雄性BALB / c小鼠，所述管道排出从胰腺的尾部外分泌酶结扎而器官的头部，位于邻近所述胃和十二指肠，不受影响。年龄，性别和体重匹配的雄性BALB / c小鼠进行假手术扼要所有步骤局部结扎的手术，除了胰管的结扎。胰腺组织收获3，7，14，后30天手术。 当PDL进行正确执行，小鼠出现健康和不显示在体重一个显著差异（ 图1A）或血糖（ 图1B）相比，假手术小鼠。外分泌腺腺泡组织逐渐丧失的PDL手术（ 图2A-E）后，导致P的连接部的小型化和重量<html> DL胰腺（ 图3），从关于此称为PDL尾巴。三天后的PDL，腺泡组织形态变得破碎和腺泡细胞凋亡（ 图4），并有可能通过渗透CD45 +免疫细胞（ 图5）吞噬。在后第7天的PDL，许多腺泡小叶由纤维化（ 图6）和脂肪组织（ 图2C-E），而其余的腺泡细胞经历腺泡到导管化生取代。在第14天后的PDL，几乎所有的腺泡lobuli是缺乏腺泡细胞（ 图2A-E）的制造中的PDL尾胰腺出现半透明的，使得胰岛变得可见，以肉眼（ 图3）。一致腺泡细胞凋亡的开始时，增加的导管上皮细胞周期活动观察（ 图7）。 PDL引起的增加胰岛素+β细胞体积 内容】"&gt;两周的PDL后在PDL中尾的总β细胞体积比非结扎深水尾了一倍。β细胞量是通过测量INS +区域4微米的部分量化，36微米间隔，横跨整个组织占总胰腺体积的10％。PDL诱导如可以示出通过自动整-tissue光学断层摄影（OPT）。15由于β细胞大小的PDL后不改变手术相比，非结扎胰腺两周后增加胰岛素含量增加β细胞量是增加β细胞数的结果。 PDL导致增加β细胞的增殖和Ngn3的表达激活 β细胞增殖免疫组织化学（IHC）染色分析胰腺中收获7天14天假或PDL手术后。 β细胞阳性增殖标记物Ki-67的的百分比通过在量化spection单个细胞在非自动化方式。在第7和14天后的PDL手术，在PDL中尾的β细胞增殖显著相比增加非结扎胰腺。 内PDL中的胚胎胰岛祖标记相比，非结扎胰腺中的PDL尾Ngn3的是显著增加的表达后3天。 Ngn3的成绩单最高水平1周内达成，随后缓慢下降。我们经常测量Ngn3的基因激活表达的PDL胰腺相对于十二指肠稳定Ngn3的水平Ngn3的编码mRNA的水平。最近，我们建议，新生的PDL中的程度可能取决于Ngn3的基因活化的水平。15,30 <img alt="图1" src="/files/ftp_upload/52765/52765fig1.jpg" /> 图1. PDL不影响体重和血糖。体重（G）（A）和血糖（毫克/ dL）的（B）中从假手术（白色条）和PDL小鼠（黑色条），测量在不同时间点（第0天，第7，第14和30），在手术后，并没有表现出假和PDL之间的任何显著差异手术小鼠在任何时间点。图最初由Xu 等出版，2008年11。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52765/52765fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52765/52765fig2.jpg" /> 图2. PDL导致腺泡细胞的逐渐丧失 。由苏木精-伊红染色，在第3天（B），7（C），14（D）和30（E）手术后显露PDL胰腺尾部相比，假手术胰尾（A）的形态变化。 3天后结扎，腺泡组织只有一个微妙的破坏可为Observed（B）中 。 7天后结扎，腺泡组织被严重破坏和导管配合已经形成（C）。 14天结扎后，一些腺泡细胞保持和lobuli通过导管结构取代腺泡，纤维网和脂肪细胞（与面板C和E星号（*）表示）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52765/52765fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。 。</a> <img alt="图3" src="/files/ftp_upload/52765/52765fig3.jpg" /> 图3. PDL尾巴在收获。深水尾（A）和PDL尾（B）中收获的第14天手术后。 PDL尾巴是大大减小尺寸相比深水尾巴。图象（C）示出的PDL尾巴在14天手术后在原位 。由于腺泡细胞流失，PDL尾出现半透明的，主胰管清晰可见（用黑色箭头指示）和小岛是可见的白色小点（用白色箭头表示）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52765/52765fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52765/52765fig4.jpg" /> 图4. PDL诱导的腺泡细胞凋亡。在3,7和14天的PDL尾免疫染色裂解-胱天蛋白酶3手术后揭示凋亡小体中的腺泡隔室在第3天以及7天后手术，而腺泡细胞来自几乎不存在PDL尾巴14天倍率条是25微米。图最初由Xu 等出版，2008年11。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52765/52765fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt<html> ="图5"SRC ="/文件/ ftp_upload / 52765 / 52765fig5.jpg"/&gt; 图5. PDL诱导免疫细胞浸润。免疫染色的白细胞标记物CD45，呈现出高数量的免疫细胞中的PDL尾部存在手术后第7天相比假胰腺。放大倍率酒吧50微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52765/52765fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52765/52765fig6.jpg" /> 图6. PDL引起的纤维化。免疫染色α-平滑肌肌动蛋白显示了一个纤维网络的PDL尾部存在手术后14天相比，假胰腺。放大倍率酒吧50微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52765/52765fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="&lt;/ HTML">"图7"SRC ="/文件/ ftp_upload / 52765 / 52765fig7.jpg"/&gt; 图7. PDL引起的增加导管的细胞增殖。双重免疫染色的导管细胞标记细胞角蛋白19（CK19）和增殖标志物的BrdU示出了增加在管细胞的细胞周期的活性。放大倍率酒吧25微米。图最初由Xu 等出版，2008年11。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52765/52765fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation at JoVE.co

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

通过胰管结扎手术损伤的小鼠胰腺作为模型内分泌和外重新规划和扩散

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

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Universidad San Sebastian

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16.1K 次观看.  Vrije Universiteit Brussel. This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation. 

视频: Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

自体胰腺癌小鼠模型研究不可逆电穿孔的影响

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

科研

癌症研究

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.

Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

三维细胞培养中胰腺肌囊内增生的模拟与操纵

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

毛尿胰腺腺腺腺癌超声引导正射植入

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

胰腺癌矫形小鼠模型

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.

Syngeneic mouse orthotopic allografts of pancreatic ductal adenocarcinoma (PDAC) recapitulate the biology, phenotypes, and therapeutic responses of disease subtypes. Owing to their fast, reproducible tumor progression, they are widely used in preclinical studies. Here, we show common practices to generate these models, injecting syngeneic murine PDAC cultures into the pancreas.

同系小鼠原位同种异体移植物模拟胰腺癌

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, ...

<meta charset="utf8"></head><body>在胰腺癌模型中使用肿瘤浸润淋巴细胞的过继治疗

Flow Cytometry-Based Isolation and Therapeutic Evaluation of Tumor-Infiltrating Lymphocytes in a Mouse Model of Pancreatic Cancer

Pancreatic cancer is an aggressive malignancy with a dismal prognosis and limited therapeutic options. Adoptive cell therapy, which involves isolating and activating a patient&#39;s own immune cells, such as tumor-infiltrating lymphocytes (TILs), before re-infusing them, represents a promising experimental approach. However, techniques for adoptive cell transfer in preclinical pancreatic cancer models are not well established. Here, we describe a detailed protocol for adoptive cell therapy using TILs from a syngeneic pancreatic cancer mouse model. The procedure involves implanting live or irradiated mouse pancreatic cancer cells in fluorescence-labeled reporter mice to initiate immune cell influx, then isolating lymphocytes from primary tumors via flow cytometry sorting and/or activating and expanding tumor-reactive T cells ex vivo, and adoptively transferring these activated T cells intraperitoneally into tumor-bearing mice, followed by interleukin-2 administration.&#160;Bioluminescent tumor imaging allows for longitudinal monitoring of orthotopic tumor growth and response to therapy, especially evaluating the tumor-specific cytotoxic effects. This approach recapitulates the logistics involved in developing adoptive cell transfer therapies for pancreatic cancer patients. The results demonstrate enhanced antitumor efficacy of adoptively transferred tumor-reactive T cells compared to irrelevant lymphocyte controls. This versatile methodology enables the in vivo study of adoptive immunotherapy in pancreatic cancer as well as the optimization of cell processing parameters and combination treatment regimens.

<meta charset="utf8"></head><body>基于流式细胞术的胰腺癌小鼠模型中肿瘤浸润淋巴细胞的分离和治疗评估

We describe a method to utilize tumor-infiltrating lymphocytes (TILs) from mice through flow cytometry for adoptive cell transfer. This protocol aims to verify the specific cytotoxicity of TILs against tumors in a syngeneic pancreatic cancer mouse model, providing insights into the development of adoptive cell therapies for pancreatic cancer.

基于流式细胞术的胰腺癌小鼠模型中肿瘤浸润淋巴细胞的分离和治疗评估

Pancreatic cancer is an aggressive malignancy with a dismal prognosis and limited therapeutic options. Adoptive cell therapy, which involves isolating and ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

应用改良袖套技术的小鼠胰腺移植模型

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

医学

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

解剖的小鼠胰腺的组织学分析与代谢谱

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

通过胰管结扎手术损伤的小鼠胰腺作为模型内分泌和外重新规划和扩散

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此视频中的章节

自体胰腺癌小鼠模型研究不可逆电穿孔的影响

三维细胞培养中胰腺肌囊内增生的模拟与操纵

毛尿胰腺腺腺腺癌超声引导正射植入

胰腺癌矫形小鼠模型

同系小鼠原位同种异体移植物模拟胰腺癌

基于流式细胞术的胰腺癌小鼠模型中肿瘤浸润淋巴细胞的分离和治疗评估

基于流式细胞术的胰腺癌小鼠模型中肿瘤浸润淋巴细胞的分离和治疗评估

基于流式细胞术的胰腺癌小鼠模型中肿瘤浸润淋巴细胞的分离和治疗评估

应用改良袖套技术的小鼠胰腺移植模型

解剖的小鼠胰腺的组织学分析与代谢谱