Name: retrograde parotid gland infusion through stensen's duct in a non-human primate for vectored gene delivery
Uploaded: 2021-08-12T14:00:00
Description: Watch this Scientific Journal Video about Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery at JoVE.com

作者要感谢卡格尼·盖特先生在拍摄过程中的视听支持。我们还要感谢赫夫纳VA医疗中心为追求这个项目提供学术支持。

所有程序都在威克森林医学院克拉克森校区进行动物研究。当局动物护理及使用委员会（IACUC）已征询有关合乎道德的考虑，并提交审查程序的细节。威克森林国际自然保护联盟批准了我们的研究协议，所有程序都是根据IACUC批准的协议#A17-147完成的。
1. 准备输液装置
<ol><li>使用一把剪刀将尺寸 10 聚乙烯管 （PET10） 切割成 25 厘米长。</li>
	<li>使用黑色标记在 1 厘米和 2 厘米?...

<ol>
	<li>Isenman, L., Liebow, C., Rothman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretion+of+mammalian+digestive+enzymes+by+exocrine+glands.">The secretion of mammalian digestive enzymes by exocrine glands.</a> The American Journal of Physiology. 276, 223-232 (1999).</li><li>Perez, 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=Salivary+epithelial+cells:+An+unassuming+target+site+for+gene+therapeutics.">Salivary epithelial cells: An unassuming target site for gene therapeutics.</a> The International Journal of Biochemistry &amp; Cell Biology. 42, 773-777 (2010).</li><li>Kochel, T. 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=A+dengue+virus+serotype-1+DNA+vaccine+induces+virus+neutralizing+antibodies+and+provides+protection+from+viral+challenge+in+Aotus+monkeys.">A dengue virus serotype-1 DNA vaccine induces virus neutralizing antibodies and provides protection from viral challenge in Aotus monkeys.</a> Vaccine. 18, 3166-3173 (2000).</li><li>Ponzio, T. A., Sanders, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+salivary+gland+as+a+target+for+enhancing+immunization+response.">The salivary gland as a target for enhancing immunization response.</a> Tropical Diseases, Travel Medicine and Vaccines. 3, 4 (2017).</li><li>Baum, B. 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=Early+responses+to+adenoviral-mediated+transfer+of+the+aquaporin-1+cDNA+for+radiation-induced+salivary+hypofunction.">Early responses to adenoviral-mediated transfer of the aquaporin-1 cDNA for radiation-induced salivary hypofunction.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 19403-19407 (2012).</li><li>Voutetakis, 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=Sorting+of+Transgenic+Secretory+Proteins+in+Rhesus+Macaque+Parotid+Glands+After+Adenovirus-Mediated+Gene+Transfer.">Sorting of Transgenic Secretory Proteins in Rhesus Macaque Parotid Glands After Adenovirus-Mediated Gene Transfer.</a> Human Gene Therapy. 19, 1401-1405 (2008).</li><li>Niedzinski, E. 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=Enhanced+systemic+transgene+expression+after+nonviral+salivary+gland+transfection+using+a+novel+endonuclease+inhibitor/DNA+formulation.">Enhanced systemic transgene expression after nonviral salivary gland transfection using a novel endonuclease inhibitor/DNA formulation.</a> Gene Therapy. 10, 2133-2138 (2003).</li><li>Niedzinski, E. 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=Zinc+Enhancement+of+Nonviral+Salivary+Gland+Transfection.">Zinc Enhancement of Nonviral Salivary Gland Transfection.</a> Molecular Therapy. 7, 396-400 (2003).</li><li>Samuni, Y., Baum, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+in+salivary+glands:+From+the+bench+to+the+clinic.">Gene delivery in salivary glands: From the bench to the clinic.</a> Biochimica et Biophysica Acta - Molecular Basis of Disease. , (2011).</li><li>Voutetakis, 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=Adeno-Associated+Virus+Serotype+2-Mediated+Gene+Transfer+to+The+Parotid+Glands+of+Nonhuman+Primates.">Adeno-Associated Virus Serotype 2-Mediated Gene Transfer to The Parotid Glands of Nonhuman Primates.</a> Human Gene Therapy. 18, 142-150 (2007).</li></ol>

在这里，我们描述了通过斯滕森的管道向帕罗蒂德腺体进行逆行输注的协议。所描述的方法提供了指导，研究人员可以探索唾液组织作为基因治疗和其他应用场所的效用。
有许多关键步骤可以确保程序的成功。首先，所有程序步骤都应轻轻完成。有力的支撑嘴可能导致气管下行。强制罐装的帕皮拉或快速注入溶液到斯滕森的管道可能导致急性导管撕裂或慢性导管狭窄。
<p ...

虽然唾液腺以产生唾液的外分泌而闻名，但研究人员早已认识到它们能够将蛋白质直接分泌到血液中，从而成为系统性施用（如替代激素或抗体生产）基因治疗的潜在靶点。事实上，唾液腺比其他组织靶点具有几个优势，例如产生分泌蛋白质的固有能力（缺乏特性肌肉）、可以限制病媒扩散的重封装，以及为不整合载体提供稳定性的分化良好的组织。此外，在发生严重不良事件时，唾液腺对生命不至关重要，可以手术切除。虽然不是立即直观，帕罗蒂腺也很容易从嘴里通过他们的主要排泄管道，斯滕森的管道2。
鉴于唾液组织对基因治疗的益处，人们越来越有兴趣探索这一组织目标。在啮齿动物、犬类和非人类灵长类动物模型中已经进行了许多研究，至少一项人类临床试验正在进行中。为了进一步探索和发展这种组织靶点用于基因治疗的目的，将需要更多的非人类灵长类动物研究。本文描述了一种通过斯滕森的管子访问帕罗蒂腺的方法，以提供一个向量基因，用于在非人类灵长类动物模型中进行转导。为了明显地演示输液和管道进入腺体的解剖学，使用放射性对照器进行了荧光镜检查。为了证明一个载体的成功转导，使用了腺病毒血清型5（Ad5）矢量egfp基因。Ad5 是一种描述良好的载体，能够转导唾液组织。虽然它太免疫原性，最终临床使用，Ad5载体被选中为这个示范研究，以确保有效的转导。评估增强型绿色荧光蛋白 （EGFP） 的生产是一种经过充分描述的方法，可以证明转导后向量基因的转录和翻译成功，并在此完成。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>500 &#181;L U100 syringes with 30-gauge needles</td>
			<td>Becton Dickinson</td>
			<td>328466</td>
			<td>fixed needle for less waste</td>
		</tr>
		<tr>
			<td>Adhesive (e.g., Ethicon Dermabond)</td>
			<td>Various</td>
			<td></td>
			<td>Cyanoacrylate adhesive to seal and keep the tubing in the duct during infusion.</td>
		</tr>
		<tr>
			<td>Atropine injectable solution</td>
			<td>Patterson Veterinary</td>
			<td>07 869-6061</td>
			<td>Atropine inj. 0.54 mg/mL</td>
		</tr>
		<tr>
			<td>BD Ultra-Fine Insulin Syringes 30G</td>
			<td>Walmart</td>
			<td>N/A</td>
			<td>Avilable in 0.5 mL and 1.0 mL sizes.</td>
		</tr>
		<tr>
			<td>Cyanoacrylate (medical glue)</td>
			<td>Ethicon</td>
			<td>DNX12</td>
			<td>Dermabond topical skin adhesive</td>
		</tr>
		<tr>
			<td>Dental loops with light</td>
			<td>Amazon (DDP)</td>
			<td>B012M3IV80</td>
			<td>Used to enhance visualization of Stensen&#39;s duct papilla</td>
		</tr>
		<tr>
			<td>Infant Lacrimal Dilator</td>
			<td>Surgipro</td>
			<td>SPOI-137</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine injectable solution</td>
			<td>Patterson Veterinary</td>
			<td>07-803-6637</td>
			<td>Ketaset inj. 100 mg/mL</td>
		</tr>
		<tr>
			<td>Lacrimal Dilator</td>
			<td>Surgipro</td>
			<td>SPOI-132</td>
			<td>Used to dialate the Stensen&#39;s duct.</td>
		</tr>
		<tr>
			<td>Midazolam injectable solution</td>
			<td>Patterson Veterinary</td>
			<td>07 890-6698</td>
			<td>Midazolam inj. 5mg/mL</td>
		</tr>
		<tr>
			<td>Pair of scissors</td>
			<td>Amazon (DDP)</td>
			<td>N/A</td>
			<td>Used to cut PET10 tube</td>
		</tr>
		<tr>
			<td>Polyethylene Tubing (PE-10)</td>
			<td>Scientific Comodities, Inc</td>
			<td>BB31695-PE/1</td>
			<td>Tubing connecting the 30G syringe and inserted into the duct.</td>
		</tr>
		<tr>
			<td>Q-tips</td>
			<td>Walmart</td>
			<td>N/A</td>
			<td>Used to spread cyanoacrylate on the cheek</td>
		</tr>
		<tr>
			<td>Size 10 Polyethylene Tube (PET 10)</td>
			<td>Scientific Commodities</td>
			<td>BB31695-PE/1</td>
			<td>low density polyethylene tubing</td>
		</tr>
		<tr>
			<td>Small Animal Mouth Opener</td>
			<td>Amazon (DDP)</td>
			<td>B01F3LVJXC</td>
			<td>Used to keep the animal&#39;s mouth open.</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Amazon (DDP)</td>
			<td>N/A</td>
			<td>Used to insert PET10 tube into Stenson&#39;s duct</td>
		</tr>
		<tr>
			<td>Zinc Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7646-86-7</td>
			<td>Included in plasmid DNA infusates</td>
		</tr>
	</tbody>
</table>

retrograde parotid gland infusion through stensen's duct in a non-human primate for vectored gene delivery

唾液腺是基因治疗的有吸引力的组织靶点，其有希望的结果已经导致人体试验。它们天生就能够将蛋白质分泌到血液中，并且很容易获得，因此它们有可能成为替代激素生产或通过基因转移接种疫苗的优越组织部位。建议的基因传递方法包括皮下注射和通过唾液导管逆行输液。我们演示如何在非人类灵长类动物中执行逆行唾液格兰输液 （RSGI）。我们描述了重要的解剖学地标，包括鉴定帕皮拉，一种利用基本牙科工具、聚乙烯管和氰酸丙烯酸酯对斯滕森的导管进行罐装和密封的创伤性方法，以及适当的输液率。虽然这是创伤最小的分娩方法，但该方法仍然受到能够交付的体积（<0.5 mL）以及管道和腺体可能遭受创伤的限制。我们使用荧光镜检查证明，注入物可以完全输送到腺体中，并通过免疫造血术进一步证明典型载体的转导和传递基因的表达。

成功的程序、转录和转录 图1 显示了后高级脸颊上与第 2摩尔相邻的帕罗蒂帕皮拉。图像还显示了嘴支撑的正确位置，硬味觉上的橡胶端和虹膜犬上的另一个橡胶端。 图2 显示了在PET10上2厘米标记下成功吃到帕罗蒂帕皮拉后拍摄的图像。 图3 显示了放射性逆向输液时的荧光镜图像，演示了溶液通过斯滕森的?...

Watch this Scientific Journal Video about Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery at JoVE.com

Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery

唾液腺已被提议为基因治疗的组织靶点，特别是在通过基因转移接种疫苗方面。我们演示了利用逆行性皮质输液的非人类灵长类动物模型中的基因传递。

逆行帕罗蒂德格兰德输液通过斯滕森的管道在非人类灵长类病原体矢量基因交付

Salivary glands are an attractive tissue target for gene therapy with promising results already leading to human trials. They are inherently capable of ...

此协议很重要，因为它详细演示了逆行唾液腺输液的程序。该程序可用于多种医疗原因，如病媒免疫预防或基因治疗。这种技术很容易学习和复制。尽管作者不是口腔外科医生，也没有这种背景，但还是多次成功地执行了这一手术。麻醉后使用牙齿百叶窗放大，并识别与上二摩尔相邻的后脸颊上的或斯滕森导管的开口。将圆锥形蒸狄机的点放入中心或打开，然后来回旋转蒸腾器约 20 到 30 秒，轻轻地拉平。接下来，使用钳子将聚乙烯管的标记端固定在含注射器的注入物上，距离远端约 0 . 5 厘米，然后轻轻地将管尖插入扩张的。通过小旋转动作轻轻推进管子，以帮助管子滑动，然后调整钳子 0.5 厘米接近以前的抓地力。重复此过程，直到两厘米标记达到帕罗蒂帕皮拉。接下来，要密封帕皮拉，减少注入物在口腔中的溢出，在周围和插入管子上涂抹青藻。一旦氰酸酯干燥，慢慢注射超过五分钟，以每分钟100微升的速度。输液完成后，将管子留在原位至少五分钟，从而保持管道密封，并允许输液留在帕罗蒂德腺体中。最后，用轻柔的牵引力取出管子，氰化物将随管子一起拉出。使用无线电对比输液的荧光镜检查显示溶液通过斯滕森的导管分枝并进入帕罗蒂腺。免疫作为对皮罗蒂腺的化学分析显示，在导管细胞和阴道细胞中EGFP的阳性染色表明两种细胞类型的成功转导和转录。这个程序的主要挑战可能是将聚乙烯管引入乳腺，这应该非常温和和缓慢地完成。

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在极少数情况下，表皮转谷氨酰胺酶IgA和组织转谷氨酰胺酶2抗体的识别恒河猴皮炎

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唾液，唾液腺，淋巴收集肩突硬蜱蜱

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