三维细胞培养中胰腺肌囊内增生的模拟与操纵
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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。 高压灭菌聚丙烯网方块, 以及两对剪刀和钳子。 制作100毫升的 10倍 waymouth 溶液。将 waymouth 的粉…
Representative Results
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…
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 37 °C shaking incubator | Thermo Scientific | SHKE4000-7 | |
| 5% CO2, 37 °C Incubator | NUAIRE | NU-5500 | |
| 50 ml tubes | Falcon | 352070 | |
| Absorbent pad, 20" x 24" | Fisherbrand | 1420662 | |
| Adenovirus, Ad-GFP | Vector Biolabs | 1060 | |
| Aluminum foil | Fisherbrand | 01-213-101 | |
| BD Precision Glide Needle 21G x 1/2 | Fisher Scientific | 305167 | Use as pins for dissection |
| Beaker, 600 mL | Fisherbrand | FB-101-600 | |
| Bleach, 8.25% | Clorox | 31009 | |
| Cell Recovery Solution | Corning | 354253 | Referred to as 'basement membrane matrix recovery solution' in the manuscript. |
| Centrifuge | Beckman Coulter | Allegra X-15R Centrifuge | |
| Collagenase Type I, Clostridium histolyticum | Sigma | C0130-1G | 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 °C. |
| Dexamethasone | Sigma | D1756 | Create a 4 mg/ml solution by dissolving powder in methanol, aliquoting and storing at -20 °C. |
| Ethanol, 200 proof | Decon Laboratories | 2701 | |
| Fetal Bovine Serum | Sigma | F0926-100mL | |
| Forceps | Fine Science Tools | 11002-12 | |
| Forceps | Fine Science Tools | 91127-12 | |
| Glass slide, 8-well | Lab-Tek | 177402 | |
| Hank's Balanced Salt Solution (HBSS), No calcium, No magnesium, No phenol red | Fisher Scientific | SH3048801 | |
| Ice bucket, rectangular | Fisher Scientific | 07-210-103 | |
| Instant Sealing Sterilization Pouch | Fisherbrand | 01-812-51 | |
| LAB GUARD specimen bags (for mouse after dissection) | Minigrip | SBL2X69S | |
| Lentiviral Packaging Mix, Virapower | Invitrogen | 44-2050 | |
| Matrigel | Corning | 356234 | Referred to as 'basement membrane matrix' in the manuscript. |
| Parafilm | Bemis | PM992 | Referred to as 'plastic paraffin film' in the manuscript. |
| PBS | Fisher Scientific | SH30028.02 | |
| Penicillin-Streptomycin | ThermoFisher Scientific | 15140122 | |
| Pipet tips, 10 µl | USA Scientific | 1110-3700 | |
| Pipet tips, 1000 µl | Olympus Plastics | 24-165RL | |
| Pipet tips, 200 µl | USA Scientific | 1111-1700 | |
| Pipet-Aid | Drummond | ||
| PIPETMAN Classic P10, 1-10 μl | Gilson | F144802 | |
| PIPETMAN Classic P1000, 200-1000 μl | Gilson | F123602 | |
| PIPETMAN Classic P20, 2-20 μl | Gilson | F123600 | |
| PIPETMAN Classic P200, 20-200 μl | Gilson | F123601 | |
| Pipettes, 10 ml | Falcon | 357551 | |
| Pipettes, 25 ml | Falcon | 357525 | |
| Pipettes, 5 ml | Falcon | 357543 | |
| Plate, 12-well | Corning Costar | 3513 | |
| Plate, 24-well plate | Corning Costar | 3524 | |
| Plate, 35 mm | Falcon | 353001 | |
| Plate, 6-well | Falcon | 353046 | |
| Polybrene | EMD Millipore | TR-1003-G | Create a 40 mg/ml solution by dissolving powder in sterile molecular biology grade water, aliquoting and storing at -20 °C. |
| Polypropylene Mesh, 105 µm | Spectrum Labs | 146436 | |
| Polypropylene Mesh, 500 µm | Spectrum Labs | 146418 | |
| Scissors | Fine Science Tools | 14568-12 | |
| Scissors | Fine Science Tools | 91460-11 | |
| Sodium Bicarbonate (Fine White Powder) | Fisher Scientific | BP328-500 | |
| Sodium Hydroxide | Fisher Scientific | S318-500 | |
| Soybean Trypsin Inhibitor 8 | Gibco | 17075029 | |
| Spatula | Fisherbrand | 21-401-10 | |
| Steriflip 50 ml, 0.22 micron filters | Millipore | SCGP00525 | |
| TGF-α | R&D Systems | 239-A-100 | |
| Type I Rat Tail Collagen | Corning | 354236 | |
| Waymouth MB 752/1 Medium (powder) | Sigma | W1625-10X1L | |
| Weigh boat, hexagonal, medium | Fisherbrand | 02-202-101 |
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Citazione di questo articolo
Fleming Martinez, A. K., Storz, P. Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture. J. Vis. Exp. (144), e59096, doi:10.3791/59096 (2019).