我们想感谢 Gundula Hertl 的支持。

本议定书的所有程序均经德国动物福利法和准则批准。考虑使用 10-到12周的 NMRI 裸鼠和 C57Bl/6 小鼠 (捐赠者: 老女性; 收件人: 男性) 贯穿整个实验。使用 NMRI 裸鼠为胰岛隔离。根据当地动物设施规定, 将小鼠置于规定的条件下。
1. 链脲佐菌素诱导糖尿病
<ol><li>4天, 用称重机测量10至12周的 NMRI 裸鼠和 C57Bl/6 小鼠的体重。</li>
	<li>根据测量的体重, 准备180毫克/千克剂量的链脲佐菌素在0.1 毫升生理盐水每只老鼠。 注: 链脲佐菌素应在使用和覆盖铝箔之前进行新的准备, 因为它是光敏的。</li>
	<li>注射自体和同种异体小鼠每一个剂量的0.1 毫升链脲佐菌素 (180 毫克/千克) 腹腔使用26克针。</li>
	<li>日-3,-2 和-1 收集2-4 µL 的血液通过穿刺的尾部静脉的非禁食小鼠。</li>
	<li>用每条试纸的2-4 µL 测定 NMRI 裸鼠和 C57Bl/6 小鼠的血糖 注意: 在这一点上, 80% 的老鼠应该成为糖尿病 (20 毫摩尔/升)。<ol>
<li>在胰岛移植时使用小鼠血糖水平达到20毫摩尔/升。</li>
	</ol>
</li>
</ol>2. 小鼠胰岛的分离
<ol><li>麻醉小鼠 (100 毫克/千克体重) 和甲苯噻嗪 (20 毫克/千克体重) 腹腔注射与26克针。 注: 氯胺酮和甲苯噻嗪的体积是根据动物的体重 (0.10 mL/10 g) 确定的。</li>
	<li>保持小鼠仰卧位, 用聚维酮碘溶液进行消毒。通过检查踏板反射来确认正确的安乐死。 注: 万一 C57Bl/6 剃了腹部区域。</li>
	<li>用细钳握住皮肤, 用剪刀做中线切口, 露出腹腔。将肠道放置在不育纱布压缩下的腹腔左侧和外腔。</li>
	<li>调整胃在另一个方向, 以找到胰腺和其他相关组织。</li>
	<li>到达主动脉, 做一个小切口排泄血液。使用无菌纱布进行血液吸收。</li>
	<li>小心地取出肠道。将胰腺与胃和脾脏分开。</li>
	<li>把胰脏放在新鲜的培养皿里。用镊子取出所有不需要的材料, 如脂肪。</li>
	<li>用4毫升的胶原酶填充5毫升的注射器, 用26克针 (股票溶液: 在汉克溶液的12毫升中用30毫克胶原酶) 注入胰腺。</li>
	<li>用剪刀将胰腺剁碎, 增加表面积, 改善消化, 转移到12毫升红帽管。 注: 汉克的平衡盐溶液: HBSS 10x (100 毫升), 青霉素-链霉素 (10 毫升), 庆大霉素 (1 毫升), 环丙沙星 (10 毫升), HEPES (35 毫升), 蒸馏 H2O (900 毫升)</li>
	<li>在颤抖的水浴中进行10分钟的消化, 37 摄氏度。在十五年代的固定时间间隔内, 对样品进行三次涡旋。</li>
	<li>用手大力混合消化的胰腺3分钟, 并将其放在冰上十年代。</li>
	<li>添加6毫升的冷汉克溶液, 以停止消化反应和离心机在 560 x g 在室温下3分钟。</li>
	<li>在室温下取出上清液, 在10毫升的帕克/胎小牛血清 (P/FCS) 培养基中溶解颗粒。 注: P/FCS 介质: TCM-199 10x (100 毫升), FCS (50 毫升), 青霉素-链霉素 (10 毫升), HEPES (20 毫升), 蒸馏 H2O (900 毫升)</li>
	<li>根据光镜下的形态学 (球状, 金黄色-褐色) 识别胰岛。避免带深色中心的小岛。在光显微镜下进行双硫腙染色, 检查纯度和活性。 注: 双硫腙与锌离子结合, 为胰岛提供红色染色。</li>
	<li>用1毫升的吸管在光显微镜下计数和精选胰岛, 将它们转移到含有4毫升 P/FCS 培养基的新鲜培养皿 (60 毫米 x 15 毫米)。 注: 培养皿中的P/FCS 介质的体积不应超过4毫升, 以确保氧气的可用性。</li>
	<li>将孤立的胰岛在4毫升的 P/FCS 培养基中过夜, 在37摄氏度的孵化器。</li>
</ol>3. 胰岛移植动物的制备
<ol><li>从37摄氏度孵化器中取出孤立的小岛, 并将它们放在流动工作台下。中心的胰岛与一个圆形摆动运动的培养皿。</li>
	<li>用0.1 毫升的 P/FCS 培养基填充1毫升注射器 (蓝色23克针)。然后, 在0.3 毫升的汉克溶液中添加350个胰岛。将注射器保持在垂直位置, 使小岛能够安定下来。</li>
	<li>将体积减小到0.05 毫升, 用褐色的26克针取代蓝色的23克针。对同一注射器, 增加0.15 毫升的 Ficoll (密度梯度培养基) 到顶端, 以达到最终体积0.2 毫升的注射器。避免气泡。</li>
	<li>将注射器保持在垂直位置, 稍微旋转, 以避免胰岛结块。胰岛将在2-3 分钟内在注射器的锥形处沉淀。</li>
	<li>测量受体小鼠的体重和血糖水平;血糖水平应约为20毫摩尔/升。</li>
	<li>麻醉小鼠 (100 毫克/千克体重) 和甲苯噻嗪 (20 毫克/千克体重) 腹腔注射26克针。</li>
	<li>将小鼠置于仰卧位, 放在暖热板上 (37 摄氏度)。通过捏脚趾来检查麻醉深度。</li>
	<li>用聚维酮碘溶液 (皮肤消毒剂) 清洁腹部区域。为防止眼部干燥, 请用眼膏保湿。 注: 万一 C57Bl/6 剃了腹部区域。</li>
</ol>4. 胰岛移植通过门静脉路线
<ol><li>0天, 从剖腹手术开始, 用钳夹住皮肤, 用剪刀做2-3 厘米长的中线切口。将无菌湿纱布放在伤口边缘周围, 保持湿润。</li>
	<li>将肠道置于左侧和腹腔外的无菌纱布压缩。 注意: 在整个过程中保持肠道湿润, 预热前0.9% 氯化钠溶液或不育纱布浸泡在生理盐水中。</li>
	<li>移动十二指肠并定位胰腺。通过将胰腺向上推, 找到位于腹侧部位的门静脉。使用手指和拇指暴露的门静脉, 一旦暴露, 刺穿它附近的肝脏。</li>
	<li>在1分钟内, 在放大镜的检查下缓慢和稳步地将胰岛 (0.2 毫升从步骤 3.4) 注入门静脉, 以避免胰岛的任何渗漏。 注: 注射后, 应始终检查注射器中的其余胰岛。在注射器中服用1毫升的 P/FCS 介质, 冲洗成新鲜的培养皿, 并在显微镜下计数。</li>
	<li>在穿刺部位放置一个不育的量规, 用食指 (穿透深度: 0.5-1 厘米) 施加压力, 以6分钟止血。 注: 注射量不应超过0.2 毫升。由于剪切应力, 体积和压力过大会破坏小岛。</li>
	<li>小心地将肠道、腹膜、腹部肌肉层和筋膜放回原位。</li>
	<li>使用镊子, 抬起皮肤, 用可吸收的丝缝线闭合肌肉切口, 上部皮肤层有2-3 个夹子。</li>
	<li>用皮肤消毒剂清洁表面, 应用伤口治疗, 并将老鼠放在单独的笼子里。伤口治疗提供了耐水性丙烯酸酯层, 防止浸渍和继发感染。 注意: 观察老鼠, 直到他们恢复足够的意识, 提供曲马多 (每只老鼠0.2 毫克) 连同液体。在本协议中, 小鼠快速从麻醉中恢复, 曲马多管理减轻了疼痛。7天后伤口就会愈合</li>
	<li>用普通的食物和水喂老鼠。食品和水的消耗和尿液排泄将增加后, 佐菌素注射液。</li>
	<li>每天在自体小鼠的第一周和每周一次测量血糖水平。在同种异体小鼠中, 每周三次测量血糖水平长达15天。</li>
</ol>

<ol>
	<li>McCall, M., Shapiro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+islet+transplantation.">Update on islet transplantation.</a> Cold Spring Harbor Perspectives in Medicine. 2 (7), a007823 (2012).</li><li>Shapiro, A. 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=Islet+transplantation+in+seven+patients+with+type+1+diabetes+mellitus+using+a+glucocorticoid-free+immunosuppressive+regimen.">Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.</a> New England Journal of Medicine. 343 (4), 230-238 (2000).</li><li>Cantarelli, E., 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=Bone+marrow+as+an+alternative+site+for+islet+transplantation.">Bone marrow as an alternative site for islet transplantation.</a> Blood. 114 (20), 4566-4574 (2009).</li><li>Echeverri, G. J., 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=Endoscopic+gastric+submucosal+transplantation+of+islets+(ENDO-STI):+technique+and+initial+results+in+diabetic+pigs.">Endoscopic gastric submucosal transplantation of islets (ENDO-STI): technique and initial results in diabetic pigs.</a> American Journal of Transplantation. 9 (11), 2485-2496 (2009).</li><li>Korsgren, O., 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=Optimising+islet+engraftment+is+critical+for+successful+clinical+islet+transplantation.">Optimising islet engraftment is critical for successful clinical islet transplantation.</a> Diabetologia. 51 (2), 227-232 (2008).</li><li>Merani, S., Toso, C., Emamaullee, J., Shapiro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+implantation+site+for+pancreatic+islet+transplantation.">Optimal implantation site for pancreatic islet transplantation.</a> British Journal of Surgery. 95 (12), 1449-1461 (2008).</li><li>Lingwal, 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=Inhibition+of+gelatinase+B+(matrix+metalloprotease-9)+activity+reduces+cellular+inflammation+and+restores+function+of+transplanted+pancreatic+islets.">Inhibition of gelatinase B (matrix metalloprotease-9) activity reduces cellular inflammation and restores function of transplanted pancreatic islets.</a> Diabetes. 61 (8), 2045-2053 (2012).</li><li>Chen, 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=Improved+intraportal+islet+transplantation+outcome+by+systemic+IKK-beta+inhibition:+NF-kappaB+activity+in+pancreatic+islets+depends+on+oxygen+availability.">Improved intraportal islet transplantation outcome by systemic IKK-beta inhibition: NF-kappaB activity in pancreatic islets depends on oxygen availability.</a> American Journal of Transplantation. 11 (2), 215-224 (2011).</li><li>Wang, Y., Wang, T., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+aspects+are+critical+for+carrying+out+intraportal+islet+transplants+successfully+in+a+diabetes+mouse+model.">Three aspects are critical for carrying out intraportal islet transplants successfully in a diabetes mouse model.</a> Experimental and Clinical Transplantation. 10 (3), 302-304 (2012).</li><li>Neuman, J. C., Truchan, N. A., Joseph, J. W., Kimple, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+mouse+pancreatic+islet+isolation+and+intracellular+cAMP+determination.">A method for mouse pancreatic islet isolation and intracellular cAMP determination.</a> Journal of Visualized Experiments. (88), e50374 (2014).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> Journal of Visualized Experiments. (50), (2011).</li><li>Rother, K. I., Harlan, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+facing+islet+transplantation+for+the+treatment+of+type+1+diabetes+mellitus.">Challenges facing islet transplantation for the treatment of type 1 diabetes mellitus.</a> Journal of Clinical Investigation. 114 (7), 877-883 (2004).</li><li>Su, 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=Small+islets+are+essential+for+successful+intraportal+transplantation+in+a+diabetes+mouse+model.">Small islets are essential for successful intraportal transplantation in a diabetes mouse model.</a> Scandinavian Journal of Immunology. 72 (6), 504-510 (2010).</li><li>Rekittke, N. E., Ang, M., Rawat, D., Khatri, R., Linn, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+Therapy+of+Type+1+Diabetes+Mellitus:+From+Pancreatic+Islet+Transplantation+to+Mesenchymal+Stem+Cells.">Regenerative Therapy of Type 1 Diabetes Mellitus: From Pancreatic Islet Transplantation to Mesenchymal Stem Cells.</a> Stem Cells International. 2016, 22 (2016).</li></ol>

所有作者都宣称他们没有利益冲突。

本文对胰岛移植的门静脉路线进行了探讨。该协议在缓解糖尿病应激方面具有高效性, 为探索胰岛移植研究提供了深入的机会。该协议证实, 大约350只小岛能够逆转高血糖状态。此外, 所有的老鼠在手术过程中都没有死亡, 所有接受者的血糖控制都达到了。定期记录体重和血糖水平, 以反映移植胰岛逆转高血糖的能力、标准和功能。在 syngeneically 移植的小鼠中, 血糖水平每天在第一周和一周内测量一?...

胰岛移植是治疗1型糖尿病的一种很有前景的方法1。第一次尝试转移绵羊胰腺碎片到糖尿病患者是由沃森威廉斯在1893年进行。然而, 通过埃德蒙顿协议实现了临床胰岛移植的一个重大突破, 此后又制定了一系列国家计划2。过去, 在临床前模型中探讨了骨髓、网膜囊、肌内区、胃粘膜表面、脾、肾胶囊等几个部位, 但门静脉系统被认为是适宜有效的部位之一。对于临床程序3,4,5,6。
胰岛素的外源性管理可以用胰岛输液代替门静脉。这被认为是一个首选的网站, 因为氧气供应是可比的, 在当地的胰腺由于在下游的位置的汇合的门动脉和静脉。此外, 还有一个很大的表面积, 这样可以保留胰岛的三维结构, 并且可以方便地进行血管化5。在接受糖尿病的老鼠中, 2000 人或猪胰岛当量或350小鼠胰岛足以逆转高血糖状态7,8。钳水平被报告了15天在小鼠接受者模型的异种胰岛和在同种异体鼠-鼠模型, 并在120天的自体鼠对鼠移植。
与通过门静脉胰岛移植的疗效相关的因素是适当的麻醉、穿刺方法和止血9。麻醉可以诱导吸入 (5% 异氟烷) 或注射 (氯胺酮, 甲苯噻嗪, 或戊巴比妥), 这些物质往往结合。为了控制麻醉的深度和时间, 应注意鼠标的状态, 如粘膜的颜色、眼盖、角膜反射、呼吸速率和体温。重要的是要确保动物不挣扎, 它生存的行动。温度可由不同的加热装置维护, 如加热垫、红光灯泡、等.热热板已用于保持25-30 摄氏度之间的温度, 在鼠标和操作表之间, 防止发生低温。麻醉药的剂量是重要的, 因为所有这些药物都是由肝脏代谢的, 肝功能可能短暂紊乱的胰岛悬浮液注入.门静脉的理想穿刺点是第一和第二支流静脉的位置。
胰岛的数量和预期的注射量也会影响移植的结果, 因为过量的体积可能会增加剪切应力。0.2 毫升体积被认为适合胰岛移植入门静脉。穿刺伤口 (26 口径针) 诱导门静脉出血, 需要及时有效地停止。无菌纱布或手指可在穿刺部位施加最小压力, 以止血6分钟左右。结合, 门胰岛注射液是有效的, 并提供调节血糖水平的化学诱导糖尿病小鼠模型。

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="20">
			<td>C57Bl/6 mice&#160;</td>
			<td>Charles River (Sulzfeld, Germany)</td>
			<td></td>
			<td>(10-12 weeks)</td>
		</tr>
		<tr height="20">
			<td>BALB/c mice</td>
			<td>Charles River (Sulzfeld, Germany)</td>
			<td></td>
			<td>(10-12 weeks)</td>
		</tr>
		<tr height="20">
			<td>NMRI nude mice</td>
			<td>Charles River (Sulzfeld, Germany)</td>
			<td></td>
			<td>(10-12 weeks)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketamine</td>
			<td>Bela-pharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylazine</td>
			<td>CEVA TIERGESUNDHEIT GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe, 23G and 26G needle</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri Dish</td>
			<td>Falcon</td>
			<td>303800</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Carl Roth</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Gauze</td>
			<td>Fuhrmann</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Shaking Water bath</td>
			<td>Kotterman</td>
			<td>1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td>Braun</td>
			<td>1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose Meter&#160;</td>
			<td>One Touch</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Warm Thermal Plate</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Braunol (povidone-iodide solution)</td>
			<td>B.Braun Melsungen AG</td>
			<td>3864154</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liposic Edo - Sprinkled Ointment (eye ointment)</td>
			<td colspan="2">Dr. G Mann Chem.-Pharm.&#160; GmbH</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubator</td>
			<td>Fa. Roth</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Flow Bench</td>
			<td>Herdus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ficoll</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-0 Silk Suture Vicryl</td>
			<td>Braun</td>
			<td></td>
			<td>(7.5 *1.75mm)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Michel Clamps (clips)</td>
			<td>Aesculap&#160;</td>
			<td>BN507R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">P/FCS Solution</td>
			<td>Gibco</td>
			<td>21180-021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TCM-199 (10x)</td>
			<td>Gibco</td>
			<td>21180-021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FCS</td>
			<td>Biowest</td>
			<td>S1810-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (1 M)</td>
			<td>Biochrom</td>
			<td>L 1613</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamycin</td>
			<td>Ratiopharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ciprofloxacin</td>
			<td>Fresenius Kabi</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hank&#39;s Solution</td>
			<td>Gibco</td>
			<td>14060-040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase</td>
			<td>Roche</td>
			<td>11213865001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptozotocin</td>
			<td>Calbiochem</td>
			<td>572204</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OPSITE-spray (wound healer)</td>
			<td>Smith and nephew</td>
			<td>66004978</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Reugular food</td>
			<td>Altromin</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Head light LED 16042 (magnifying glass)</td>
			<td>Eschenbach</td>
			<td>16042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat anti-mouse PECAM-1(CD31) monoclonal primary antibody</td>
			<td>Millipore, Chemicon</td>
			<td>CBL1337</td>
			<td>Dilution (1:100)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-rat secondary antibody</td>
			<td>Dianova</td>
			<td>712095153</td>
			<td>Dilution (1:400)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyclonal guinea pig anti-insulin primay antibody</td>
			<td>Dako</td>
			<td>A0564</td>
			<td>Dilution (1:500)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-guinea pig secondary antibody</td>
			<td>Dianova</td>
			<td>706-259-148</td>
			<td>Dilution (1:400)</td>
		</tr>
	</tbody>
</table>

intraportal transplantation of pancreatic islets in mouse model

胰岛移植降低高血糖是非常成功的啮齿动物与化学性糖尿病。实验性胰岛移植最常见的移植部位为肾囊。然而, 由于对胰岛与血液成分的相互作用知之较少, 因此在实验性胰岛移植中利用门静脉途径也有意义。
该协议表明了门静脉胰岛移植技术在 NMRI 裸鼠。腹腔注射佐菌素 (180 毫克/千克) 诱导受体小鼠高血糖。他们被认为是糖尿病在非空腹血糖水平大于20毫摩尔/升。在移植前的某一天, 用胶原酶消化法从供体胰腺中分离出小鼠胰岛;每位糖尿病患者至少使用350个胰岛。根据胰岛的分离率, 每个受体使用两个或更多的捐献小鼠。在隔夜文化在37°c 以后, 胰岛通过门静脉被管理入接受者肝脏。手术后, 小鼠被保护在红色 Makrolon 的房子和观察, 直到是清醒的。该协议维持血糖控制120天的自体小鼠和15天的同种异体小鼠。

研究了化学诱导的糖尿病小鼠移植胰岛的能力。胰岛移植通过门静脉系统的糖尿病小鼠模型。捐助者接受率为2:1。在自体小鼠模型中, 利用两只捐献小鼠获得350个胰岛。然后将胰岛移植到糖尿病小鼠体内。移植后1天观察到血糖水平的降低, 提示移植胰岛在降低血糖水平中的作用。链脲佐菌素增加了血糖水平高达约22毫摩尔/升。胰岛移植后, 血糖水平下降到4.6 毫摩尔/升 (1 天...

Watch this Scientific Journal Video about Intraportal Transplantation of Pancreatic Islets in Mouse Model at JoVE.com

Intraportal Transplantation of Pancreatic Islets in Mouse Model

胰岛移植是实现1型糖尿病血糖浓度的一种途径。本文主要研究了通过门静脉途径维持糖尿病小鼠正常血糖水平的移植技术。

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 ...

intraportal-transplantation-of-pancreatic-islets-in-mouse-model

Research

JoVE Journal

Medicine

9.4K 次观看.  Clinical Research Lab. 胰岛移植是实现1型糖尿病血糖浓度的一种途径。本文主要研究了通过门静脉途径维持糖尿病小鼠正常血糖水平的移植技术。

视频: Intraportal Transplantation of Pancreatic Islets in Mouse Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

科研

医学

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States1. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available2. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat3 and later transferred to the mouse4. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease5,6. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis2,7. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system8, hormones9,10 and environmental factors11,12 affect endometriosis as well as the effects of endometriosis on fertility13 and pain14.

A description of the surgical induction of endometriosis in mice and rats by auto-transplantation of uterine tissue to the arterial cascade of the intestinal mesentery.

自动子宫组织移植手术诱发子宫内膜异位症小鼠模型

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of ...

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

人类胰岛染色协议

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

部分原位肝移植大鼠模型的肝动脉重建的外科手术

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

从手术标本的人类胰岛细胞激光显微切割的改进方法

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

肺动脉瓣使用异位心脏移植小鼠模型移植

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

瘦素受体<sup&gt;分贝</sup&gt; 2型糖尿病小鼠模型:胰岛分离和活细胞双光子成像完好胰岛

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

原位后肢移植中的鼠标

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

套筒技术在主动脉移植小鼠模型中的应用-教学视频

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

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.

胰岛门静脉移植小鼠模型的研究

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