利用定制设计的趋电钱伯斯研究前列腺细胞定向迁移
Summary
We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.
Abstract
生理电场供应特定的生物功能，如指导细胞迁移在胚胎发育，神经元向外生长和上皮伤口愈合。 体外施加直流电场到培养的细胞诱导定向细胞迁移，或趋电。我们在这里展示的2维趋电方法经修改以定做的聚（氯乙烯）（PVC）的腔室，玻璃表面，铂电极和使用在其上的细胞成像的机动阶段。在PVC室和铂电极具有低毒性，且价格合理和可重复使用的。在玻璃表面和电动显微镜阶段改善图象的质量，并允许可能的修改，以在玻璃表面，并处理该细胞。我们拍摄的两个非致瘤性，SV40永生化前列腺细胞系，PRNS-1-1和PNT2的趋电。这两种细胞系表现出类似的迁移速度和迁移均朝阴极，但它们确实显示出不同程度的方向性的趋电。通过此协议中得到的结果表明，PRNS-1-1和PNT2细胞株可具有管其定向洄游响应不同的内在特征。
Introduction
内源性电场在各种组织，如皮肤1，32，33和脑2检测到。生理电场供应特定的生物功能，包括引导胚胎发育3，第4，指导神经元突起5,6的向外生长，促进上皮细胞和角膜伤口闭合1,7。 在体外 ，施加直流电场来培养细胞模仿生理电场和诱导定向细胞迁移，或趋电。趋电已经研究中的成纤维细胞8，鱼角化9，人上皮和角膜角化10-12，淋巴细胞13，神经母细胞2和神经元祖细胞14。当暴露于施加的场，大部分研究细胞的定向迁移朝向阴极（ – ）极。然而，一些癌症的细胞，包括高转移人乳腺癌细胞和人前列腺癌细胞系PC-3M，移动到anodal（+）极15,16。提出调解趋电或解释的细胞，以感测电场的能力，包括活化几种机制的表皮生长因子受体12，上皮钠通道17，PI3K和PTEN的18，和钙离子释放15，19，其机制还没有完全理解，这是可能的，多种信号传导途径中涉及趋电。
表征粘附，游动细胞的定向迁移，既可以监视单个细胞迁移10，12，17或迁移融合细胞的片材18，20，该技术是由改性后，我们在这里展示的2维趋电的方法是有用鹏贾菲21，和西村等 10与定制，透明PVC室，可移动coverslIPS允许趋电性进行二次分析，如免疫荧光成像后容易细胞检索。的趋电室中的玻璃表面的光兼容，这使得拍摄在高放大倍率和与荧光标记的细胞。它也允许在实验设计与玻璃表面的改性，如改变表面涂层或收费。制成的第1号盖玻片隔件被用于在腔室，以最小化电流的流动过的细胞;因此焦耳热，这是成比例的电流的平方，将不会在实验过程中过热的细胞。连接琼脂桥防止与细胞中的电极的直接接触，并防止在趋电介质的pH值或离子浓度的改变。
两个非致瘤性的人类前列腺细胞系检查在这项研究中的趋电响应。该PRNS-1-1 22和PNT223顷既SV40永生化，生长因子依赖性细胞系的表达上皮标志物细胞角蛋白5，8，18和19与前列腺特异性抗原（PSA）的低或无表达。两种细胞系维持正常上皮细胞的多边形形态，但染色体异常中观察到核型22，24，虽然PRNS-1-1和PNT2在大多数实验中有着相似的行为，但它们确实显示出在腺泡结构的形成，并在不同趋电性。在3-D矩阵，基底膜，将PRNS-1-1细胞形成空心腺泡结构，类似于流明的正常前列腺腺体组织25。然而，PNT2细胞形成固体球体无管腔或极化上皮26。所述PRNS-1-1细胞也表现出比在目前的研究中PNT2更高趋电响应。形成腺泡结构和趋电之间的相关性PRNS-1-1表明趋电信号可在组织PR发挥作用ostate腺组织的运动响应于内源性电场，并提供了进一步的特征，这些2细胞系之间进行区分。
Protocol
1.培养前列腺细胞培养PRNS-1-1和PNT2前列腺细胞上100毫米培养皿在RPMI 1640培养基补充有10％FBS和抗生素-抗真菌剂，在37℃，5％的CO 2。每天刷新培养基，直到细胞达到80％汇合的趋电实验。 2.装配趋电钱伯斯组装底部室擦拭塑料趋电室用2-丙醇。适用的海洋级硅密封剂周围的圆形开口与注射器的腔室的底侧，并附加一个大玻璃罩（ 图1）。</…
Representative Results
两行前列腺细胞（PRNS-1-1和PNT2）进行了调查用这种方法。在这两个细胞系在超过2小时（ 图5A）的过程类似的速度为1.0 +/- 0.3微米/分钟迁移。然而，方向性的电场是0.7 +/- 0.3的PRNS-1-1线，和0.2±0.8对PNT2线（ 图5B）。结果显示在这两个细胞系的趋电一显著差异（p <0.01，100个细胞被跟踪），这表明它们具有导致不同的定向响应位置线索不同细胞信号传导机制。 <p class="jove_c…
Discussion
一个细胞的趋电性反应的分析，一直是许多细胞迁徙或增长的重要功能指标处理27，28。在这里，我们使用一个特制室玻璃表面拍摄2前列腺细胞系。这些细胞系表现出不同程度的趋电的，和我们推测，细胞内定位或趋电介导蛋白的激活可能产生不死的细胞系，从而导致在趋电响应的观察到的差异的过程中受到干扰。
相比在培养皿27，28作出其它趋电室的设计中?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
前列腺细胞系敬请凌钰王博士和幸吉恩孔博士在癌症中心，加州大学戴维斯分校提供的。该项目由美国国立卫生研究院资助趋电性支持4R33AI080604。
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Cells | |||
| pRNS-1-1 prostate cells | Lee et. al., (1994) | ||
| PNT2 prostate cells | Sigma-Aldrich | 95012613-1VL | Berthon et al., (1995) |
| Medium and solutions | |||
| RPMI 1640 medium | Invitrogen | 11875-093 | warm up to 37°C before use |
| Fetal Bovine Serum – Premium | Atlanta Biologicals | S11150 | 10% in PBS, warm up to 37°C before use |
| Antibiotic-Antimycotic (100x) | Life Technologies | 15240 | add 5mL to 500mL medium |
| 2-propanol | VWR | BDH1133-5GL | |
| PBS | – | – | 137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4 and 1.5mM KH2PO4 in 1000mL of H2O, pH to 7.4 and autoclaved, warm up to 37°C before use |
| 0.25% Trypsin-EDTA | Invitrogen | 25200-056 | warm up to 37°C before use, treat cells for 3-5 min at 37°C |
| Galvanotaxis device | |||
| Galvanotaxis chambers | Precision Plastics Inc, CA | custom-designed (1/4" X 2" X 3.5"), non-toxic, clear PVC chambers | |
| Galvanotaxis electrodes | UCD electric shop | platinum coiled electrodes with flexable cords | |
| Galvanotaxis power box | Substrate Engineering, CA | custom-designed DC power output with voltmeter | |
| Microscope Cover Glass, Large, 45X50mm-No. 1.5 | Fisher | 12-544-F | |
| Microscope Cover Glass, small, 25X25mm, No. 1 | ThermoScientific | 3307 | |
| Diamond point marker | ThermoScientific | 750 | |
| Marine grade silicon sealer, clear | 3M | 051135-08019 | |
| High vacuum grease | Dow Corning | 2021846-0807 | |
| 6mL syringe | Fisher Scientific | 05-561-64 | |
| Nichiryo Syringe, 1.5mL | Nichiryo | SG-M | |
| Cotton applicators | Purtian Medical Products | 806-WC | |
| Qtips | Johnson & Johnson | 729389 | |
| Nalgene 180 PVC tubing | Nalgene | 8000-9030 | 503/16 ID x 5/16 OD x 1/16 Wall |
| Bacto-Agar | Difco | 0140-01 | make 2% agar solution |
| Razor Blade | Personna | 74-0001 | |
| Equipments and Software | |||
| Benchtop Centrifuge | Eppendorf | 5810R | operated with an A-4-62 rotor |
| Cellometer Auto T4 | Nexcelom | Auto T4 | |
| Cellometer counting chambers | Nexcelom | CHT4-SD100-002 | load 20uL cell solutions to count |
| Culture Temp Warming plate | Bel-Art Scienceware | 370150000 | to keep the galvanotaxis chambers at 37°C |
| Eclipse TE-2000 microscope with motorized stage and environmental chamber | Nikon | ||
| Plan Fluor 10X/0.30 objective len | Nikon | ||
| Retiga EX CCD camera | Qimaging | Cooled CCD camara, mono-color, 12-bit | |
| Compressed air with 5% CO2 | Airgas | special order | |
| Volocity 6.3 | PerkinElmer | Image acquiring software | |
| Improvision OpenLab 5.5.2 | PerkinElmer | Cell tracking software and customized to measure migration angles | |
| FileMaker Pro Advanced, 8.0 | FileMaker | ||
| Microsoft Excel 2008 for Mac | Microsoft |
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Cite This Article
Yang, H., Dinh La, T., Isseroff, R. R. Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells. J. Vis. Exp. (94), e51973, doi:10.3791/51973 (2014).