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>

<ol>
	<li>Shaw, J. E., Sicree, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+estimates+of+the+prevalence+of+diabetes+for.">Global estimates of the prevalence of diabetes for.</a> Diabetes Res Clin Pract. 87, 4-14 (2010).</li><li>Danaei, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National,+regional,+and+global+trends+in+fasting+plasma+glucose+and+diabetes+prevalence+since+1980:+systematic+analysis+of+health+examination+surveys+and+epidemiological+studies+with+370+country-years+and+2&#183;7+million+participants.">National, regional, and global trends in fasting plasma glucose and diabetes prevalence since 1980: systematic analysis of health examination surveys and epidemiological studies with 370 country-years and 2&#183;7 million participants.</a> Lancet. 378, 31-40 (2011).</li><li>Borowiak, M., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+beta+cells.">How to make beta cells.</a> Current opinion in Cell Biology. 21, 727-732 (2009).</li><li>Gradwohl, G., Dierich, A., LeMeur, M., Guillemot, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=neurogenin3+is+required+for+the+development+of+the+four+endocrine+cell+lineages+of+the+pancreas.">neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas.</a> Proc Natl Acad Sci U S A. 97, 1607-1611 (2000).</li><li>Gu, G., Dubauskaite, J., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+for+the+pancreatic+lineage:+NGN3++cells+are+islet+progenitors+and+are+distinct+from+duct+progenitors.">Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors.</a> Development. 129, 2447-2457 (2002).</li><li>Dor, Y., Brown, J., Martinez, O. I., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+pancreatic+beta-cells+are+formed+by+self-duplication+rather+than+stem-cell+differentiation.">Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation.</a> Nature. 429, 41-46 (2004).</li><li>Teta, M., Rankin, M. M., Long, S. Y., Stein, G. M., Kushner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+regeneration+of+adult+beta+cells+does+not+involve+specialized+progenitors.">Growth and regeneration of adult beta cells does not involve specialized progenitors.</a> Dev Cell. 12, 817-826 (2007).</li><li>Bonner-Weir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspective:+Postnatal+pancreatic+beta+cell+growth.">Perspective: Postnatal pancreatic beta cell growth.</a> Endocrinology. 141, 1926-1929 (2000).</li><li>Brennand, K., Huangfu, D., Melton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+beta+cells+contribute+equally+to+islet+growth+and+maintenance.">All beta cells contribute equally to islet growth and maintenance.</a> PLoS Biol. 5, e163 (2007).</li><li>Salpeter, S. J., Klein, A. M., Huangfu, D., Grimsby, J., Dor, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+and+aging+control+the+quiescence+period+that+follows+pancreatic+beta+cell+replication.">Glucose and aging control the quiescence period that follows pancreatic beta cell replication.</a> Development. 137, 3205-3213 (2010).</li><li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta+cells+can+be+generated+from+endogenous+progenitors+in+injured+adult+mouse+pancreas.">Beta cells can be generated from endogenous progenitors in injured adult mouse pancreas.</a> Cell. 132, 197-207 (2008).</li><li>Wang, R. N., Kloppel, G., Bouwens, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duct-+to+islet-cell+differentiation+and+islet+growth+in+the+pancreas+of+duct-ligated+adult+rats.">Duct- to islet-cell differentiation and islet growth in the pancreas of duct-ligated adult rats.</a> Diabetologia. 38, 1405-1411 (1995).</li><li>Yasuda, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+expression+and+induction+of+acinar+cell+apoptosis+after+pancreatic+duct+ligation+in+mice.">Cytokine expression and induction of acinar cell apoptosis after pancreatic duct ligation in mice.</a> Journal of interferon, &amp; cytokine research : the official journal of the International Society for Interferon and Cytokine Research. 19, 637-644 (1999).</li><li>Van Gassen, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+dynamics+are+regulated+by+local+macrophage+proliferation+and+monocyte+recruitment+in+injured+pancreas.">Macrophage dynamics are regulated by local macrophage proliferation and monocyte recruitment in injured pancreas.</a> European journal of immunology. , (2015).</li><li>Van de Casteele, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenin+3++cells+contribute+to+beta-cell+neogenesis+and+proliferation+in+injured+adult+mouse+pancreas.">Neurogenin 3+ cells contribute to beta-cell neogenesis and proliferation in injured adult mouse pancreas.</a> Cell Death Dis. 4, e523 (2013).</li><li>Xiao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+evidence+for+beta+cell+neogenesis+in+murine+adult+pancreas.">No evidence for beta cell neogenesis in murine adult pancreas.</a> The Journal of clinical investigation. 123, 2207-2217 (2013).</li><li>Ackermann Misfeldt, A., Costa, R. H., Gannon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-cell+proliferation,+but+not+neogenesis,+following+60%+partial+pancreatectomy+is+impaired+in+the+absence+of+FoxM1.">Beta-cell proliferation, but not neogenesis, following 60% partial pancreatectomy is impaired in the absence of FoxM1.</a> Diabetes. 57, 3069-3077 (2008).</li><li>Lee, C. S., De Leon, D. D., Kaestner, K. H., Stoffers, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+pancreatic+islets+after+partial+pancreatectomy+in+mice+does+not+involve+the+reactivation+of+neurogenin-3.">Regeneration of pancreatic islets after partial pancreatectomy in mice does not involve the reactivation of neurogenin-3.</a> Diabetes. 55, 269-272 (2006).</li><li>Bonner-Weir, S., Sharma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+stem+cells.">Pancreatic stem cells.</a> The Journal of pathology. 197, 519-526 (2002).</li><li>Nir, T., Melton, D. A., Dor, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+from+diabetes+in+mice+by+beta+cell+regeneration.">Recovery from diabetes in mice by beta cell regeneration.</a> The Journal of clinical investigation. 117, 2553-2561 (2007).</li><li>Bouwens, L., Rooman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+pancreatic+beta-cell+mass.">Regulation of pancreatic beta-cell mass.</a> Physiol Rev. 85, 1255-1270 (2005).</li><li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurog3+gene+dosage+regulates+allocation+of+endocrine+and+exocrine+cell+fates+in+the+developing+mouse+pancreas.">Neurog3 gene dosage regulates allocation of endocrine and exocrine cell fates in the developing mouse pancreas.</a> Developmental biology. 339, 26-37 (2010).</li><li>Pan, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+patterns+of+multipotentiality+in+Ptf1a-expressing+cells+during+pancreas+organogenesis+and+injury-induced+facultative+restoration.">Spatiotemporal patterns of multipotentiality in Ptf1a-expressing cells during pancreas organogenesis and injury-induced facultative restoration.</a> Development. 140, 751-764 (2013).</li><li>Kopp, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox9++ductal+cells+are+multipotent+progenitors+throughout+development+but+do+not+produce+new+endocrine+cells+in+the+normal+or+injured+adult+pancreas.">Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas.</a> Development. 138, 653-665 (2011).</li><li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+Neurog3+expression+in+hormone-expressing+islet+cells+is+required+for+endocrine+maturation+and+function.">Sustained Neurog3 expression in hormone-expressing islet cells is required for endocrine maturation and function.</a> Proc Natl Acad Sci U S A. 106, 9715-9720 (2009).</li><li>Kopinke, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+tracing+reveals+the+dynamic+contribution+of+Hes1++cells+to+the+developing+and+adult+pancreas.">Lineage tracing reveals the dynamic contribution of Hes1+ cells to the developing and adult pancreas.</a> Development. 138, 431-441 (2011).</li><li>Rankin, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Cells+are+not+generated+in+pancreatic+duct+ligation-induced+injury+in+adult+mice.">beta-Cells are not generated in pancreatic duct ligation-induced injury in adult mice.</a> Diabetes. 62, 1634-1645 (2013).</li><li>Solar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+exocrine+duct+cells+give+rise+to+insulin-producing+beta+cells+during+embryogenesis+but+not+after+birth.">Pancreatic exocrine duct cells give rise to insulin-producing beta cells during embryogenesis but not after birth.</a> Dev Cell. 17, 849-860 (2009).</li><li>Furuyama, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+cell+supply+from+a+Sox9-expressing+progenitor+zone+in+adult+liver,+exocrine+pancreas+and+intestine.">Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine.</a> Nat Genet. 43, 34-41 (2011).</li><li>Van de Casteele, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+duct+ligation:+beta-cell+proliferation+and+beyond.">Partial duct ligation: beta-cell proliferation and beyond.</a> Diabetes. 63, 2567-2577 (2014).</li><li>Zhang, J., Rouse, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+and+pathogenesis+of+caerulein-,+duct+ligation-,+and+arginine-induced+acute+pancreatitis+in+Sprague-Dawley+rats+and+C57BL6+mice.">Histopathology and pathogenesis of caerulein-, duct ligation-, and arginine-induced acute pancreatitis in Sprague-Dawley rats and C57BL6 mice.</a> Histology and histopathology. 29, 1135-1152 (2014).</li><li>Martinez-Romero, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epigenetic+regulators+Bmi1+and+Ring1B+are+differentially+regulated+in+pancreatitis+and+pancreatic+ductal+adenocarcinoma.">The epigenetic regulators Bmi1 and Ring1B are differentially regulated in pancreatitis and pancreatic ductal adenocarcinoma.</a> The Journal of pathology. 219, 205-213 (2009).</li><li>Prevot, P. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+ductal+transcription+factors+HNF6+and+Sox9+in+pancreatic+acinar-to-ductal+metaplasia.">Role of the ductal transcription factors HNF6 and Sox9 in pancreatic acinar-to-ductal metaplasia.</a> Gut. 61, 1723-1732 (2012).</li></ol>

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。

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			<td>Hoechst</td>
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			<td>33342</td>
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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 ...

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16.0K 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