Name: pancreatic duct infusion: an effective and selective method of drug and viral delivery
Uploaded: 2021-09-30T13:00:00
Description: Watch this Scientific Journal Video about Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery at JoVE.com

作者没有承认。浙江省卫生与计划生育委员会基金会（20146242赠款）和温州市科技局基金会（赠款Y20140248）提供了资助。

所有动物实验都得到了匹兹堡儿童医院动物研究和护理委员会和匹兹堡大学机构动物护理和使用委员会的批准。
1. 术前准备
<ol><li>通过鼻锥将吸入的异黄酮（1 - 3% 用于维护，最高 5% 用于感应）管理到适当的麻醉水平。用钳子用脚趾捏监测麻醉的充分性。缺乏反应被认为是足够的，并确保不要过度医疗，因为呼吸抑郁症可能发生。</li>
	<li>将动物放在解剖显微镜台上，腹部?...

<ol>
	<li>Gittes, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+biology+of+the+pancreas:+a+comprehensive+review.">Developmental biology of the pancreas: a comprehensive review.</a> Dev Biol. 326 (1), 4-35 (2009).</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 (10), 2447-2457 (2002).</li><li>Weinberg, N., Ouziel-Yahalom, L., Knoller, S., Efrat, S., 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=Lineage+tracing+evidence+for+in+vitro+dedifferentiation+but+rare+proliferation+of+mouse+pancreatic+beta-cells.">Lineage tracing evidence for in vitro dedifferentiation but rare proliferation of mouse pancreatic beta-cells.</a> Diabetes. 56 (5), 1299-1304 (2007).</li><li>Raper, S. E., DeMatteo, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+in+vivo+gene+transfer+and+expression+in+normal+rat+pancreas.">Adenovirus-mediated in vivo gene transfer and expression in normal rat pancreas.</a> Pancreas. 12 (4), 401-410 (1996).</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=Pancreatic+cell+tracing,+lineage+tagging+and+targeted+genetic+manipulations+in+multiple+cell+types+using+pancreatic+ductal+infusion+of+adeno-associated+viral+vectors+and/or+cell-tagging+dyes.">Pancreatic cell tracing, lineage tagging and targeted genetic manipulations in multiple cell types using pancreatic ductal infusion of adeno-associated viral vectors and/or cell-tagging dyes.</a> Nat Protoc. 9 (12), 2719-2724 (2014).</li><li>Song, Z., 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=EGFR+signaling+regulates+beta+cell+proliferation+in+adult+mice.">EGFR signaling regulates beta cell proliferation in adult mice.</a> J Biol Chem. 291 (43), (2016).</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=M2+macrophages+promote+beta-cell+proliferation+by+up-regulation+of+SMAD7.">M2 macrophages promote beta-cell proliferation by up-regulation of SMAD7.</a> Proc Natl Acad Sci U S A. 111 (13), E1211-E1220 (2014).</li><li>Laukkarinen, J. M., Van Acker, G. J., Weiss, E. R., Steer, M. L., Perides, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+acute+biliary+pancreatitis+induced+by+retrograde+pancreatic+duct+infusion+of+Na-taurocholate.">A mouse model of acute biliary pancreatitis induced by retrograde pancreatic duct infusion of Na-taurocholate.</a> Gut. 56 (11), 1590-1598 (2007).</li><li>Lichtenstein, A., 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=Acute+lung+injury+in+two+experimental+models+of+acute+pancreatitis:+infusion+of+saline+or+sodium+taurocholate+into+the+pancreatic+duct.">Acute lung injury in two experimental models of acute pancreatitis: infusion of saline or sodium taurocholate into the pancreatic duct.</a> Crit Care Med. 28 (5), 1497-1502 (2000).</li><li>Perides, G., van Acker, G. J., Laukkarinen, J. M., Steer, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+acute+biliary+pancreatitis+induced+by+retrograde+infusion+of+bile+acids+into+the+mouse+pancreatic+duct.">Experimental acute biliary pancreatitis induced by retrograde infusion of bile acids into the mouse pancreatic duct.</a> Nat Protoc. 5 (2), 335-341 (2010).</li><li>Guo, 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=Specific+transduction+and+labeling+of+pancreatic+ducts+by+targeted+recombinant+viral+infusion+into+mouse+pancreatic+ducts.">Specific transduction and labeling of pancreatic ducts by targeted recombinant viral infusion into mouse pancreatic ducts.</a> Lab Invest. 93 (11), 1241-1253 (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=Neurogenin3+activation+is+not+sufficient+to+direct+duct-to-beta+cell+transdifferentiation+in+the+adult+pancreas.">Neurogenin3 activation is not sufficient to direct duct-to-beta cell transdifferentiation in the adult pancreas.</a> J Biol Chem. 288 (35), 25297-25308 (2013).</li></ol>

本研究详细描述了胰腺导管输注背后的方法，这是一种有效的小鼠模型，用于将基因和其他分子具体而有效地输送到胰腺。各组之间程序细节的模糊性突出了标准化协议8、9、10的必要性。
有几个关键步骤的程序，从选择年轻，健康的老鼠开始。十二指肠穿孔，无论多么小和可控，是鼠标中的创伤性事件，...

胰腺是一种双功能器官，具有内分泌和外分泌成分。许多病理学可能折磨胰腺，包括糖尿病、胰腺炎和胰腺癌。这三种疾病都标志着活跃的研究领域，不仅是为了开发即时疗法，而且是为了更好地了解它们的病理生理学。
有针对性的治疗交付方法将是有益的。我们开发了胰腺导管输液技术，这是药物和病毒输送到胰腺细胞的有效选择性方法。在这个模型中，胰腺导管有选择地被封装，普通胆管被遮盖，允许专门输送到胰腺。
该技术可以使特定细胞类型的血统追踪进一步阐明对胰腺发育和病理学重要的发育路径2，3。病毒载体中的不同促进者允许细胞系的特定靶向，并允许将基因引入这些细胞系4、5。这项工作展示了在胰腺内的绿色荧光蛋白（GFP）的表达后，胰腺内的胰腺管道注入病毒载体表达GFP与虚假手术。

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			<td height="42" style="height:42px;width:288px;">Mice (25-30g, 8-12 weeks old)</td>
			<td style="width:171px;">Jackson Laboratory</td>
			<td style="width:137px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Ketofen</td>
			<td style="width:171px;">Henry Schein Inc.</td>
			<td style="width:137px;">5487</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Ethanol (70%)</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:137px;">34923</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:288px;">Sterile Saline Solution</td>
			<td style="width:171px;">Baxter Healthcare Corp.</td>
			<td style="width:137px;">2B-13-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Protective Equipment</td>
			<td style="width:171px;"></td>
			<td style="width:137px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Hair Removal Product</td>
			<td style="width:171px;"></td>
			<td style="width:137px;"></td>
			<td>Nair or trimmer</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Gauze Pads 2in x 2in</td>
			<td style="width:171px;">Fisher Healthcare</td>
			<td style="width:137px;">22-362-178</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:288px;">Needles (30.5G and 25G)</td>
			<td style="width:171px;">Becton Dicksinson and Co.</td>
			<td style="width:137px;">305106 and 305122</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:288px;">Syringe for Ketofen and Saline</td>
			<td style="width:171px;">Becton Dicksinson and Co.</td>
			<td style="width:137px;">309657</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Syringe for Pump</td>
			<td style="width:171px;">Henke Sass Wolf</td>
			<td style="width:137px;">4010.200V0</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:288px;">Infusion Pump</td>
			<td style="width:171px;">Pump Systems Inc.</td>
			<td style="width:137px;">NE-1000</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:288px;">Infusion Catheter</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:137px;">CMF31G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Curved Bulldog Clamps (2)</td>
			<td style="width:171px;">Roboz</td>
			<td style="width:137px;">RS-7439</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Arruga Forceps</td>
			<td style="width:171px;">Roboz</td>
			<td style="width:137px;">RS-5163</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Adson Forcep</td>
			<td style="width:171px;">Roboz</td>
			<td style="width:137px;">RS-5234</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:288px;">Needle Holder</td>
			<td style="width:171px;">Roboz</td>
			<td>RS-7882</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:288px;">Scissor</td>
			<td style="width:171px;">Roboz</td>
			<td>RS-5982</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:288px;">Cotton Tip Applicators</td>
			<td style="width:171px;">Physician Sales and Services</td>
			<td>22-9988</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Dissecting Microscope</td>
			<td style="width:171px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Suture</td>
			<td style="width:171px;">Owens and Minor</td>
			<td>SXMD1B402</td>
			<td></td>
		</tr>
	</tbody>
</table>

pancreatic duct infusion: an effective and selective method of drug and viral delivery

胰腺是一种双功能器官，具有内分泌和外分泌成分。许多病理学可能折磨胰腺，包括糖尿病、胰腺炎和胰腺癌。这三种疾病都标志着活跃的研究领域，不仅是为了开发即时疗法，而且是为了更好地了解它们的病理生理学。很少有工具可以进一步研究这些领域。胰腺导管输液是一种重要的技术，可用于谱系追踪、基因导入和细胞系特定靶向。该技术需要对十二指肠和安普拉的第二部分进行复杂的解剖，然后是胆管的闭塞和胰腺导管的闭合。虽然这项技术在技术上起初具有挑战性，但应用却不尽人格。各组之间程序细节的模糊性突出了制定标准协议的必要性。本工作描述了在胰腺内的绿色荧光蛋白 （GFP） 的表达后，胰腺内的病毒载体注入病毒载体，表达 GFP 与虚假手术。输液和因此表达是特定于胰腺，没有表达存在于任何其他组织类型。

通过实践和细致的手术技术，这些小鼠的成活率应在95%以上。输液一周后，预期效果应在胰腺中明显。10至12周大的小鼠被用于胰腺管输液。在这里，我们使用腺相关病毒血清类型8（AAV8）与CMV促进剂来表达绿色荧光蛋白（GFP），相比之下，一个虚假的手术。小鼠胰腺在输液7天后被收获。胰腺、脾脏和十二指肠部分沾有胰岛素抗体和霍施特。 图1 显示...

Watch this Scientific Journal Video about Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery at JoVE.com

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

胰腺导管输液是一种重要的技术，可用于谱系追踪、基因导入和细胞系特定靶向。这里介绍了用于药物和病毒输送到胰腺细胞的胰腺导管输液技术。

胰腺管输液：药物和病毒输送的有效和选择性方法

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

这种胰腺导管输液过程的总体目标是在胰腺中实现血统追踪、基因导入和细胞系特定靶向。这种方法可以帮助回答有关影响胰腺的疾病的关键问题，包括慢性胰腺炎、糖尿病和胰腺癌。首先，将麻醉小鼠置于解剖显微镜下，使其腹部位于视野的中心。用手术胶带固定动物，然后在眼睛上涂上眼药膏，防止它们干燥。接下来，用厚厚的脱毛霜覆盖鼠标的腹部。大约三分钟后，用浸泡在70%乙醇中的纱布轻轻擦去奶油，去除所有头发。最后，用70%乙醇擦拭皮肤，然后用10%的碘化物溶液去除任何污染物并消毒皮肤。要开始手术，用手术刀创建一个中线皮肤切口，从西菲德过程延伸到腰肌。然后使用 Adson 钳子，抬起腹膜，用剪刀制作一个小中线孔。沿着腹膜的中线展开切口，使其长度等于皮肤切口的长度。接下来，使用钝缩回器拉起肝脏，在暴露普通胆管后，用弯曲的斗牛犬血管夹紧它。识别十二指肠和阿鲁加钳，把它拉出来，以显示作为白点可见的十二指肠第二部分前表面。要识别胰腺导管，请按凹槽提取十二指肠，然后将 30 - 1 / 5 量表针切线放置到十二指肠上，并在其与正对立的墙壁上进行穿刺。要注入溶液，通过创建的十二指肠切除术引入连接到输液泵的导管，并沿着导管滑动，直到在容器内可见，但不会超过一厘米，以避免绕过小导管支流。用第二个弯曲的斗牛犬血管夹紧导管来固定导管。然后打开泵，将溶液的 150 微升注入管道。接下来，注射到小鼠腹膜约1.5毫升的盐水，以避免任何注入体积的损失，然后用湿润的纱布覆盖暴露的肠道，以防止干燥。输液完成后，释放固定导管并使用斗牛犬夹，轻轻将其从胰腺导管中取出。然后从普通胆管中释放夹子。通过在单层中连续给它们冲泡，并施用两剂镇痛剂中的第一剂，第二剂将在第二天给予，从而关闭腹膜和皮肤。然后将鼠标放回位于热灯下的笼子，为动物提供食物和水。观察小鼠，直到它恢复足够的意识，表现在胸骨复位。手术后七天，从安乐死动物胰腺以及脾脏、肝脏和十二指肠中排出作为对照。将整个孤立的组织浸入4%的甲醛溶液中，并在4摄氏度下过夜。一旦组织被修复，低温保护样品，在4摄氏度的4摄氏度下浸泡在30%的蔗糖溶液中过夜。经过一夜的低温保护，捕捉冻结样品在液氮和使用微原子，削减组织部分在6微米。最后，在荧光显微镜下对各部分进行成像。这里展示的是小鼠在假手术后七天从小鼠身上切除的胰腺部分的微小图像，以及携带GFP基因的AAV8胰腺导管输注。肝脏、脾脏和十二指肠从注入 AAV8 的鼠标中分离出来，没有显示任何 GFP 信号，而胰腺则显示出突出的 GFP 表达，确认胰腺在胰腺导管输注后特定于病毒的传递。掌握后，此技术可在 10 分钟内正确执行。在执行此程序时，执行仔细解剖并进行尽可能小的十二指肠切除术以引入导管非常重要。该技术开发后，为研究胰腺的研究人员研究一些困扰器官的病理铺平了道路，包括啮齿动物和大型动物模型中的慢性胰腺炎、糖尿病和胰腺癌。看完这段视频后，你应该对如何有效和有选择地注入胰腺导管有很好的了解。

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The aim of this study was to establish a standardized protocol for drug-induced sleep endoscopy (DISE) to differentiate obstruction patterns in ...

A Novel Method: Super-selective Adrenal Venous Sampling

一种新的方法: 超选择性肾上腺静脉取样

Primary aldosteronism (PA) and subclinical Cushing&#39;s syndrome (SCS) are conditions in which the adrenal glands autonomously produce excessive amounts ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

经优化的 LC/ms 方法对囊性纤维化患者 Ivacaftor 及其主要代谢物和 Lumacaftor 的高通量分析

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

External Cephalic Version: Is it an Effective and Safe Procedure?

外部脑积水版本：它是一个有效和安全的程序吗？

External cephalic version (ECV) is an effective procedure for reducing the number of cesarean sections. To date, there is no video publication showing the ...