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 signa...

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?...

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 ...

surgical-injury-to-mouse-pancreas-through-ligation-pancreatic-duct-as

Research

JoVE Journal

Medicine

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

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

补肾胶囊对糖尿病小鼠的胰岛移植

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

科研

生物学

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 ...

癌症研究

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, ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

支架支持胰岛移植糖尿病小鼠附睾脂肪垫

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

生物工程

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

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

胰岛门静脉移植小鼠模型的研究

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a lack of effective treatments in the current clinical practice. With the easy accessibility of transgenic and knockout strains and their small size, which allows minimal doses of drugs required for in vivo evaluation, a well-established experimental model in mice is preferred for AP research. Moreover, SAP induced through sodium taurocholate (TC) is currently one of the most widely used and best characterized models. This model has been investigated for novel therapies and possible molecular events during the process of AP. Here, we present the generation of an AP mouse model using sodium taurocholate and a simple homemade microsyringe. Moreover, we also provide the methodology for the subsequent histology and serological testing.

A mouse model of severe acute pancreatitis is described herein. The procedure presented here is very rapid, simple, and accessible, thereby potentially allowing the study of the molecular mechanisms and different therapeutic interventions in acute pancreatitis in a convenient way.

建立小鼠重症急性胰腺炎模型，将牛磺胆酸钠逆行注射到胆胰管中

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a ...

Acinar-to-Ductal Metaplasia Induction: A Method to Study Acinar Cell to Ductal Cell Differentiation in 3D Ex Vivo Culture

Source: Fleming Martinez, A. K. et. al. <a href="https://www.jove.com/v/59096/" target="_blank">Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture</a>. J. Vis. Exp. (2019).
This video describes the technique of differentiating pancreatic acinar cells to ductal cells in ex vivo 3D culture. This helps to understand the mechanisms regulating the acinar-to-ductal metaplasia (ADM), a key process in the early development of pancreatic cancer.

腺泡到导管化生诱导:一种在 3D 离体培养中研究腺泡细胞到导管细胞分化的方法

Source: Fleming Martinez, A. K. et. al. Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture. J. Vis. Exp. ...