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
Summary
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.
Abstract
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。
Introduction
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”>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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors acknowledge all colleagues who made this work possible; Ann Demarré, 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).
Materials
| Supplies for preparation area | |||
| Hibiscrub | Regent Medical | 5601IE5F11 | Chlorhexidin diglucon |
| Ketamin | Ceva | BE-V202526 | anesthesia |
| Rompun(2%) (Xylazin) | Bayer | BE-V041815 | anesthesia |
| Duratears | Alcon | 34335-8 | ointment for eyes |
| Razor | |||
| Supplies for surgical area | |||
| Leica Operating microscope | Leica | 10446320 | |
| Hot bead sterilizer | Fine Science Tools (FST) | 18000-45 | |
| autoclaved surgical instruments | Fine Science Tools (FST) | ||
| Recirclulating water heating pad | Gaymar Industries, Inc. | TP702 | |
| Adhesive OP-towel | BARRIER | 706500-07 | |
| OP-tape | BARRIER | 381035-00 | |
| Stella 3/5 Compresse de gaze (Sterile) | Lohmann&Rauscher International | 35968 | |
| Mini Plasco 0,9% NaCl solution | B.BRAUN | 3521680 | |
| 6-0 prolene suture | Ethicon | 8706H | |
| 4-0 polyglycol suture | Ethicon | SL-607 | |
| Supplies for recovery area | |||
| Paper bedding (paper towel) | |||
| Vetergesic | ecuPhar | BE-V342955 | |
| Materials for analysis | |||
| Taqman Ngn3 primer | Integrated DNA technologies | Mm.PT.56a.33574796.gs | |
| Taqman CycloA primer | Integrated DNA technologies | Mm.PT.39a.2.gs | |
| anti-Rabbit Cleaved Caspase 3 antibody (D175) | Cell Signaling | 5A1E | |
| anti-mouse/rat Ki67 antibody | eBioscience | 14-5698-82 | |
| monoclonal anti-actin alpha-Smooth muscle-Cy3 (mouse) | Sigma | 085K4889 | |
| anti-rat Cytokeratin 19 | DSHB | ||
| monoclonal Mouse anti-BrdU | Dako | M0744 | |
| Hoechst | Sigma | 33342 |
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