Name: mimicking and manipulating pancreatic acinar-to-ductal metaplasia in 3-dimensional cell culture
Uploaded: 2019-02-11T14:40:00
Description: Watch this Scientific Journal Video about Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture at JoVE.com

这项工作得到了从国家卫生研究院到 p s 的 r01 赠款 (ca200572) 的支持。内容完全由作者负责, 不一定代表国家癌症研究所或国家保健研究所的官方观点. 资助者在研究设计、数据收集和分析、决定出版或准备手稿。

所有的动物工作都得到了梅奥诊所 iacu 的批准。
1. 材料、溶液和三维矩阵基座的制备
<ol><li>切割500μm 和105μm 聚丙烯以76毫米正方形格成76毫米。将每个正方形折叠两半, 以创建一个较小的折叠正方形。将一个500μm 和一个105μm 的网状方块放入可高压灭菌袋中。</li>
	<li>高压灭菌聚丙烯网方块, 以及两对剪刀和钳子。</li>
	<li>
制作100毫升的 10倍 waymouth 溶液。将 waymouth 的粉...

<ol>
	<li>Rooman, I., Real, F. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+ductal+adenocarcinoma+and+acinar+cells:+a+matter+of+differentiation+and+development.">Pancreatic ductal adenocarcinoma and acinar cells: a matter of differentiation and development.</a> Gut. 61 (3), 449-458 (2012).</li><li>Stanger, B. Z., Hebrok, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+identity+in+pancreas+development+and+regeneration.">Control of cell identity in pancreas development and regeneration.</a> Gastroenterology. 144 (6), 1170-1179 (2013).</li><li>Caldas, C., Kern, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=K-ras+mutation+and+pancreatic+adenocarcinoma.">K-ras mutation and pancreatic adenocarcinoma.</a> International Journal of Pancreatology. 18 (1), 1-6 (1995).</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=Identification+of+Sox9-dependent+acinar-to-ductal+reprogramming+as+the+principal+mechanism+for+initiation+of+pancreatic+ductal+adenocarcinoma.">Identification of Sox9-dependent acinar-to-ductal reprogramming as the principal mechanism for initiation of pancreatic ductal adenocarcinoma.</a> Cancer Cell. 22 (6), 737-750 (2012).</li><li>Morris, J. P. t., Cano, D. A., Sekine, S., Wang, S. C., Hebrok, 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-catenin+blocks+Kras-dependent+reprogramming+of+acini+into+pancreatic+cancer+precursor+lesions+in+mice.">Beta-catenin blocks Kras-dependent reprogramming of acini into pancreatic cancer precursor lesions in mice.</a> Journal of Clinical Investigation. 120 (2), 508-520 (2010).</li><li>Grippo, P. J., Nowlin, P. S., Demeure, M. J., Longnecker, D. S., Sandgren, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preinvasive+pancreatic+neoplasia+of+ductal+phenotype+induced+by+acinar+cell+targeting+of+mutant+Kras+in+transgenic+mice.">Preinvasive pancreatic neoplasia of ductal phenotype induced by acinar cell targeting of mutant Kras in transgenic mice.</a> Cancer Research. 63 (9), 2016-2019 (2003).</li><li>Sawey, E. T., Johnson, J. A., Crawford, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase+7+controls+pancreatic+acinar+cell+transdifferentiation+by+activating+the+Notch+signaling+pathway.">Matrix metalloproteinase 7 controls pancreatic acinar cell transdifferentiation by activating the Notch signaling pathway.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (49), 19327-19332 (2007).</li><li>Liou, G. Y., 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-secreted+cytokines+drive+pancreatic+acinar-to-ductal+metaplasia+through+NF-kappaB+and+MMPs.">Macrophage-secreted cytokines drive pancreatic acinar-to-ductal metaplasia through NF-kappaB and MMPs.</a> Journal of Cell Biology. 202 (3), 563-577 (2013).</li><li>De La, O. 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=Notch+and+Kras+reprogram+pancreatic+acinar+cells+to+ductal+intraepithelial+neoplasia.">Notch and Kras reprogram pancreatic acinar cells to ductal intraepithelial neoplasia.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (48), 18907-18912 (2008).</li><li>Habbe, 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=Spontaneous+induction+of+murine+pancreatic+intraepithelial+neoplasia+(mPanIN)+by+acinar+cell+targeting+of+oncogenic+Kras+in+adult+mice.">Spontaneous induction of murine pancreatic intraepithelial neoplasia (mPanIN) by acinar cell targeting of oncogenic Kras in adult mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (48), 18913-18918 (2008).</li><li>Liou, G. Y., 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=Mutant+KRas-Induced+Mitochondrial+Oxidative+Stress+in+Acinar+Cells+Upregulates+EGFR+Signaling+to+Drive+Formation+of+Pancreatic+Precancerous+Lesions.">Mutant KRas-Induced Mitochondrial Oxidative Stress in Acinar Cells Upregulates EGFR Signaling to Drive Formation of Pancreatic Precancerous Lesions.</a> Cell Report. 14 (10), 2325-2336 (2016).</li><li>Hughes, P., Marshall, D., Reid, Y., Parkes, H., Gelber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+costs+of+using+unauthenticated,+over-passaged+cell+lines:+how+much+more+data+do+we+need.">The costs of using unauthenticated, over-passaged cell lines: how much more data do we need.</a> Biotechniques. 43 (5), 577-578 (2007).</li><li>Blauer, M., Nordback, I., Sand, J., Laukkarinen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+explant+outgrowth+culture+model+for+mouse+pancreatic+acinar+cells+with+long-term+maintenance+of+secretory+phenotype.">A novel explant outgrowth culture model for mouse pancreatic acinar cells with long-term maintenance of secretory phenotype.</a> European Journal of Cell Biology. 90 (12), 1052-1060 (2011).</li><li>Gout, 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=Isolation+and+culture+of+mouse+primary+pancreatic+acinar+cells.">Isolation and culture of mouse primary pancreatic acinar cells.</a> Journal of Visualized Experiments. (78), (2013).</li><li>Artym, V. V., Matsumoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+in+three-dimensional+collagen+matrix.">Imaging cells in three-dimensional collagen matrix.</a> Current Protocols in Cell Biology. , 11-20 (2010).</li><li>Geraldo, S., Simon, A., Vignjevic, 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=Revealing+the+cytoskeletal+organization+of+invasive+cancer+cells+in+3D.">Revealing the cytoskeletal organization of invasive cancer cells in 3D.</a> Journal of Visualized Experiments. (80), e50763 (2013).</li></ol>

分离、感染和电镀原代腺细胞的时间相对较短, 是这种方法的一个优势。相反, 从外植体生长中培养腺样细胞几乎不需要动手时间, 但腺细胞13的生长需要7天。一种用于腺法分离的替代协议14指出了获取腺酰亚耳细胞的一种非常短的方法;然而, egf 是使用该协议隔离时保持腺酰亚细胞存活的重要组成部分。由于 egf 刺激腺泡细胞向导管细胞分化, 本文的方法不需要 ad...

腺管转增生 (adm) 是胰腺炎期间的一种保护机制, 也是推动胰腺癌发展的关键过程,需要进一步的机械洞察。虽然炎症引起的 adm 是可逆的 2, 致癌kras突变, 存在于90% 的胰腺癌病例3, 防止分化回阿金纳表型 4,5,6. 在三维培养中, 将原代腺细胞培养和分化为导管细胞, 可以研究调节 adm 过程的分子机制, 这在体内是很难研究的。这样的体外研究能够实时可视化 adm、其驱动程序和调节器。使用这里描述的方法确定或验证了 adm 过程的几个驱动因素, 以及对其下游信号通路的机械洞察。这些包括由 tgf-mediated (转化生长因子 alpha) 介导的 mmp-7 (基质金属蛋白酶-7) 表达诱导 adm 和激活诺奇 7, 以及 rantes (调节激活, 正常 t 细胞表达和分泌; 也已知作为趋化因子配体5或 cpl5) 和 tnfα (肿瘤坏死因子α)通过激活 nf-b (核因子-b) 8 诱导的 adm。adm 的另一个介质是致癌 kras9,10, 这导致氧化应激的上升, 通过增加 egfr (表皮生长因子受体) 及其配体的表达, egf (表皮生长因子) 和 tgf-α11。
虽然胰腺癌细胞系的使用在体外研究中很常见, 但初级细胞培养提供了许多优势。例如, 来自非转基因小鼠或具有启动突变的小鼠的原代腺细胞, 如krasg12d,是研究 adm 早期事件的最适当模型, 因为这些细胞很少有可控的突变, 这已知在胰腺癌的早期存在。这与许多胰腺癌细胞系形成鲜明对比, 这些细胞系有多种突变, 并根据第12段的内容表达不同。此外, 通过这种手段确定的信号通路已通过动物研究得到验证。使用原代腺泡细胞的一个警告是, 它们不能像典型的癌症细胞系那样转染。
本文详细介绍了腺泡细胞分离技术、通过添加兴奋剂或通过腺病毒或慢病毒感染调节蛋白表达的3d 培养技术, 以及在终点重新分离细胞进行进一步分析的技术。

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			<td height="24" style="height:24px;width:357px;">37 &#176;C shaking incubator</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:171px;">SHKE4000-7</td>
			<td style="width:1487px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:357px;">5% CO2, 37 &#176;C Incubator</td>
			<td style="width:167px;">NUAIRE</td>
			<td style="width:171px;">NU-5500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">50 ml tubes</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Absorbent pad, 20&#34; x 24&#34;</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:171px;">1420662</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Adenovirus, Ad-GFP</td>
			<td style="width:167px;">Vector Biolabs&#160;</td>
			<td style="width:171px;">1060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Aluminum foil</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:171px;">01-213-101</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">BD Precision Glide Needle 21G x 1/2</td>
			<td style="width:167px;">Fisher Scientific&#160;</td>
			<td style="width:171px;">305167</td>
			<td>Use as pins for dissection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Beaker, 600 mL</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:171px;">FB-101-600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Bleach, 8.25%</td>
			<td style="width:167px;">Clorox</td>
			<td style="width:171px;">31009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Cell Recovery Solution</td>
			<td style="width:167px;">Corning</td>
			<td style="width:171px;">354253</td>
			<td>Referred to as &#39;basement membrane matrix recovery solution&#39; in the manuscript.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Centrifuge&#160;</td>
			<td style="width:167px;">Beckman Coulter</td>
			<td style="width:171px;">Allegra X-15R Centrifuge</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Collagenase Type I, Clostridium histolyticum</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:171px;">C0130-1G</td>
			<td>Create a 100 mg/ml solution by dissolving powder in sterile molecular biology grade water. When thawing one aliquot, dilute to 10 mg/ml, filter sterilize and place 1 ml aliquots at -20 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Dexamethasone</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:171px;">D1756</td>
			<td>Create a 4 mg/ml solution by dissolving powder in methanol, aliquoting and storing at -20 &#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Ethanol, 200 proof</td>
			<td style="width:167px;">Decon Laboratories&#160;</td>
			<td style="width:171px;">2701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Fetal Bovine Serum&#160;</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:171px;">F0926-100mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Forceps&#160;</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:171px;">11002-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Forceps&#160;</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:171px;">91127-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Glass slide, 8-well</td>
			<td style="width:167px;">Lab-Tek</td>
			<td style="width:171px;">177402</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:357px;">Hank&#39;s Balanced Salt Solution (HBSS), No calcium, No magnesium, No phenol red</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:171px;">SH3048801</td>
			<td>&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Ice bucket, rectangular</td>
			<td style="width:167px;">Fisher Scientific&#160;</td>
			<td style="width:171px;">07-210-103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Instant Sealing Sterilization Pouch</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:171px;">01-812-51</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:357px;">LAB GUARD specimen bags (for mouse after dissection)&#160;</td>
			<td style="width:167px;">Minigrip</td>
			<td style="width:171px;">SBL2X69S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Lentiviral Packaging Mix, Virapower</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:171px;">44-2050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Matrigel</td>
			<td style="width:167px;">Corning</td>
			<td style="width:171px;">356234</td>
			<td>Referred to as &#39;basement membrane matrix&#39; in the manuscript.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Parafilm</td>
			<td style="width:167px;">Bemis</td>
			<td style="width:171px;">PM992</td>
			<td>Referred to as &#39;plastic paraffin film&#39; in the manuscript.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">PBS</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:171px;">SH30028.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipet tips, 10 &#181;l</td>
			<td style="width:167px;">USA Scientific</td>
			<td style="width:171px;">1110-3700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipet tips, 1000 &#181;l&#160;</td>
			<td style="width:167px;">Olympus Plastics</td>
			<td style="width:171px;">24-165RL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipet tips, 200 &#181;l&#160;</td>
			<td style="width:167px;">USA Scientific</td>
			<td style="width:171px;">1111-1700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipet-Aid</td>
			<td style="width:167px;">Drummond</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">PIPETMAN Classic P10, 1-10 &#956;l</td>
			<td style="width:167px;">Gilson</td>
			<td style="width:171px;">F144802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">PIPETMAN Classic P1000, 200-1000 &#956;l</td>
			<td style="width:167px;">Gilson</td>
			<td style="width:171px;">F123602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">PIPETMAN Classic P20, 2-20 &#956;l</td>
			<td style="width:167px;">Gilson</td>
			<td style="width:171px;">F123600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">PIPETMAN Classic P200, 20-200 &#956;l</td>
			<td style="width:167px;">Gilson</td>
			<td style="width:171px;">F123601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipettes, 10 ml&#160;</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">357551</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipettes, 25 ml</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">357525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pipettes, 5 ml&#160;</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">357543</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Plate, 12-well</td>
			<td style="width:167px;">Corning Costar</td>
			<td style="width:171px;">3513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Plate, 24-well plate</td>
			<td style="width:167px;">Corning Costar</td>
			<td style="width:171px;">3524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Plate, 35 mm</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Plate, 6-well</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:171px;">353046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Polybrene</td>
			<td style="width:167px;">EMD Millipore</td>
			<td style="width:171px;">TR-1003-G</td>
			<td>Create a 40 mg/ml solution by dissolving powder in sterile molecular biology grade water, aliquoting and storing at -20 &#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Polypropylene Mesh, 105 &#181;m</td>
			<td style="width:167px;">Spectrum Labs</td>
			<td style="width:171px;">146436</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Polypropylene Mesh, 500 &#181;m&#160;</td>
			<td style="width:167px;">Spectrum Labs</td>
			<td style="width:171px;">146418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Scissors&#160;</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:171px;">14568-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Scissors&#160;</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:171px;">91460-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Sodium Bicarbonate (Fine White Powder)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:171px;">BP328-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Sodium Hydroxide</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:171px;">S318-500</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:357px;">Soybean Trypsin Inhibitor 8</td>
			<td style="width:167px;">Gibco</td>
			<td style="width:171px;">17075029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Spatula</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:171px;">21-401-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Steriflip 50 ml, 0.22 micron filters&#160;</td>
			<td style="width:167px;">Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">TGF-&#945;</td>
			<td style="width:167px;">R&#38;D Systems</td>
			<td style="width:171px;">239-A-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Type I Rat Tail Collagen</td>
			<td style="width:167px;">Corning</td>
			<td style="width:171px;">354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Waymouth MB 752/1 Medium (powder)</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:171px;">W1625-10X1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Weigh boat, hexagonal, medium&#160;</td>
			<td style="width:167px;">Fisherbrand</td>
			<td style="width:171px;">02-202-101</td>
			<td></td>
		</tr>
	</tbody>
</table>

mimicking and manipulating pancreatic acinar-to-ductal metaplasia in 3-dimensional cell culture

在胰腺炎和胰腺癌早期发展过程中, 腺形细胞向导管细胞的分化是一个需要进一步研究的关键过程。为了了解调节腺管间增生 (adm) 的机制, 与其他系统相比, 体内三维培养和原代腺细胞向导管细胞分化具有许多优势。利用本文的技术, 蛋白质表达的调节简单而快速, 只需一天就能分离、刺激或病毒感染, 并开始培养原代腺瘤细胞来研究 adm 过程。与使用基底膜基质不同的是, 在胶原蛋白 i 细胞外基质中的腺样体细胞簇的种子, 允许腺瘤细胞在操作前保留其腺瘤的同一性。在测试各种组件对 adm 诱导的贡献时, 这一点至关重要。细胞因子或其他外代给药因素的作用不仅可以通过这种技术进行测试, 而且普通突变、蛋白质表达增加或蛋白表达的击倒的贡献也可以通过病毒感染进行测试。原代腺泡细胞, 使用腺病毒或慢病毒载体。此外, 细胞可以重新从胶原蛋白或基底膜基质在终点, 并分析蛋白的表达。

完成协议在一天内, 并在刺激下, adm 是在3-5天内看到。图 1描述了该方法的顺序, 即在第一天完成步骤1到4。这包括制剂、腺苷类分离、病毒感染和嵌入胶原蛋白或基底膜基质。当基底膜基质诱导 adm 时, 胶原蛋白中的腺形细胞需要刺激, 如 tgf-α, 以进行分化到导管细胞。第1天, 胶原蛋白或基底膜基质中的细胞具有类似葡萄的圆形外观, 如果使用病毒感染...

Watch this Scientific Journal Video about Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture at JoVE.com

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

三维细胞培养中胰腺肌囊内增生的模拟与操纵

原代腺细胞分离、蛋白表达或活性调节、培养和下游应用在腺尖囊转牙 (adm) 的体外研究中非常有用, 这是胰腺癌发展的早期事件。

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

我们的协议提供阿西纳到电图代谢的直接可视化，并允许研究异位管理因子或蛋白质调制对这个过程的贡献。该技术的主要优点是，可实时查看阿西纳到电感代谢，并可进行细胞信号研究。要解剖安乐死小鼠的胰腺，首先将小鼠的爪子固定在聚苯乙烯盖上，将其定向，使尾巴面向研究人员，用70%乙醇喷洒腹部。使用自 <3>化钳在中线抬起毛皮和皮肤，并使用自动剪剪刀，通过毛皮和皮肤从尿道开口到隔膜肋笼区域进行切口。在左侧和右侧进行额外的切口，切开毛皮和皮肤，从而清晰地查看腹腔。然后，切入内膜衬里，下到中间，到左右，把它从器官拉开。使用钳子抬起肠子并将其移到鼠标的左侧，从而创建空间，查看连接到深色、红色、椭圆形脾脏的浅粉色胰腺。胰腺组织将有柔软的海绵状纹理。切掉沿着胃跑的胰腺，与肠子交织在一起。将脾脏与胰腺分离，将胰腺放在含有 10 毫升 HBSS 的 50 毫升管中，用 1 倍青霉素链霉素将其转移到层流罩中。将管子内的东西倒入空的称重船上，用钳子用旋涡清洗胰腺。然后，使用钳子将胰腺移动到含有青霉素链霉素的 HBSS 的第二艘称重船上，然后通过旋转再次清洗。将胰腺移动到含有HBSSS的第三艘称重船，并用青霉素链霉素将胰腺切成5毫米或更小的碎片。要将称重船的内容倒入一个新的50毫升管中，首先使用钳子将胰腺碎片移入液体中，因为船正在倾斜。一旦碎片不再附着在船上，就将其内装物倒入管子中。用钳子拾起剩余的碎片，并清洗管子中的钳子。在4摄氏度的931倍重力下离心管2分钟后，使用5毫升移液器去除HSS和任何漂浮的脂肪。加入五毫升在HSSS中稀释的胶原酶。将管子包装在塑料石蜡薄膜中，以密封的盖子，确保密封的盖子。放在孵化器中，在 37 摄氏度的 220 RPM 下摇晃 20 分钟。这将产生浑浊溶液，和少数剩余的组织片断。为了停止分离，将管子放在冰上，加入5毫升的冷HBVS与5%FBS。在 4 摄氏度的 931 倍重力下离心两分钟后，使用 5 毫升移液器去除上流液。用 5%FBS 将颗粒重新悬浮在 10 毫升 HBSS 中，并在 4 摄氏度下以 931 倍重力再次离心两分钟。使用 5 毫升移液器取出上流液，然后用 5%FBS 的另外 10 毫升 HBSS 重复此步骤。然后用5%FBS将颗粒重新悬浮在五毫升的HSS中。使用 P1000 移液器通过 500 微米的网状物一次过滤一毫升细胞悬浮液，并放入 50 毫升管中。要通过网格清洗剩余的胰腺细胞，请添加额外的五毫升 HBSS 与 5%FBS。然后使用 P1000 移液器，通过 105 微米网格一次将一毫升滤液传递到 50 毫升管中。轻轻移液该细胞悬浮液到含有 HBSS 的 50 毫升管中，其 FBS 为 30%FBS，在顶部形成一层细胞悬浮液。在4摄氏度下将该电池悬浮液在233倍的重力下离心两分钟，然后取出上一代。在 Waymouth 的完整介质中重新悬浮含有 Acinar 细胞的颗粒。由于一个胰腺足以进行两种病毒感染，因此在两个35毫米板的盖子之间分离细胞悬浮液，从而允许在悬浮液中感染。对于腺病毒感染，将病毒以1/1000稀释添加到每个板的盖子和漩涡。将板放在37摄氏度和5%CO2的细胞培养箱中。第一小时每15分钟旋转一次，再持续两个小时。要制作胶原蛋白凝胶，将7毫升1型鼠尾胶原蛋白、700微升10倍于韦茅斯的介质和466.6微升的0.34摩尔氢氧化钠在50毫升的冰上管中，制作胶原蛋白凝胶。在50毫升管中，在三个小时的孵育和轻轻混合后，将等量的细胞悬浮液和胶原蛋白凝胶混合。板2000微升，一次一个井在六井板，同时保持板在冰上和漩涡均匀分布胶原蛋白细胞层。为了凝固培养基，在37摄氏度的细胞培养箱中孵育细胞培养箱，在30分钟内孵育5%的二氧化碳。然后添加1.5毫升每井1倍的威茅斯的完整介质与所需的刺激或抑制剂。第二天更改介质，然后每隔一天更改一次。根据使用的刺激，可观察到第三天至第五天之间的阿西纳到管道代谢或 ADM。感染GP腺病毒后24小时，胶原蛋白或基底膜基质中嵌入的细胞图像显示感染是有效的。在感染后五天，在胶原蛋白中嵌入的细胞在每毫升TGFα的50毫微克刺激下形成管道样结构。嵌入在地下室膜基质中的细胞在感染后五天没有刺激的情况下形成管道样结构。而较大的管道是在刺激后形成的，每毫升TGFα为50毫微克。协议中最关键的一点是胶原酶消化的长度。适当的消化由浊溶液和少数剩余的组织片段指示。

Research

JoVE Journal

Cancer Research

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

一种内在的弥漫性脑胶质瘤脑桥快速验尸细胞培养协议（DIPG）

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

科研

癌症研究

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

蒸发减少培养条件的提高多细胞球体形成的微孔板重现

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential ...

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

3维球形共培养胰腺癌细胞和成纤维细胞的制备及代谢试验

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

基于球形的头颈部鳞状细胞癌3D 细胞培养模型的治疗试验

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

从3维球体培养中分离出类似干细胞的细胞

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

监测三D培养中的癌细胞入侵和T细胞毒性

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

胰腺癌矫形小鼠模型

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

Robot Assisted Distal Pancreatectomy with Celiac Axis Resection (DP-CAR) for Pancreatic Cancer: Surgical Planning and Technique

Robot Assisted Distal Pancreatectomy with Celiac Axis Resection DP-CAR for Pancreatic Cancer: Surgical Planning and Technique

机器人协助切除腹腔轴切除术 （DP-CAR） 胰腺癌：手术规划和技术

Malignant pancreatic tumors involving the celiac artery can be resected with a distal pancreatectomy, splenectomy and celiac axis resection (DP-CAR), ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

从患者来源的肿瘤样本中建立三维微球并评估其对药物的敏感性

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...