将鼠皮皮和皮米与热解辛分离，以检测部位特异性炎症mRNA和蛋白质
Summary
这里介绍的是一个表皮与真皮分离的协议，用于评估炎症调解器的产生。炎症后，大鼠后爪表皮在4°C下由热利辛从真皮中分离出来。 然后，通过RT-PCR进行表皮分析，通过西方污点和免疫造血术进行蛋白质评估。
Abstract
在皮肤损伤、炎症和/或敏感期间，需要易于使用和廉价的技术来确定炎症调解人和神经营养素的特定产地。本研究的目标是使用热解酶（一种在4°C下活跃的蛋白酶）来描述表皮-皮肤分离协议。 为了说明这个程序，斯普拉格道利大鼠麻醉，右后爪注射卡拉吉南。注射后6小时和12小时，有炎症和天真的老鼠被安乐死，一块后爪，斑点皮肤被放置在寒冷的Dulbecco的改良鹰介质。然后，在地下室膜上用氯化钙将热碱与真皮分离。接下来，真皮由微分切钳固定，表皮被轻轻地调戏。组织部分的甲苯蓝色染色表明表皮与地下室膜上的真皮干净分离。所有角膜细胞层保持完好无损，表皮干脊以及皮肤的凹痕被清晰观察。定性和实时RT-PCR用于确定神经生长因子和间柳金-6表达水平。最终进行西式印迹和免疫造血术，以检测神经生长因子的数量。本报告表明，冷热解素消化是将表皮与真皮分离的有效方法，用于评估炎症期间的mRNA和蛋白质变化。
Introduction
从皮肤对炎症调解人和神经营养因子的评价可以受到限制，因为在发炎的真皮和表皮1，2，3中发现的细胞类型的异质性。最近对涉及两层分离或进行细胞分离评价的几种酶、化学、热或机械技术进行了审查。酸、碱、中性盐和热量可以迅速将表皮与真皮分开，但细胞外肿胀通常发生在5、6。胰腺素、弹性酶、角蛋白酶、拼贴酶、前列腺素、脱口香剂和热解酶是用于表皮-皮肤分离的酶4、7。特普辛和其他大尺度蛋白酶在37~40°C下活跃，但必须仔细监测，防止表皮层分离。迪斯帕塞在拉米纳登萨切开表皮，但在寒冷的4，8或更短的时间点在37°C 4，9中分离需要24小时。所有这些技术的一个限制特征是组织形态的潜在破坏和mRNA和蛋白质的完整性损失。
为了保持mRNA和蛋白质的完整性，应在寒冷中短期进行皮肤分离方法。在评估炎症研究的皮肤分离技术时，热解酶是一种有效的酶，在寒冷的温度下将表皮与真皮分离。热蛋白在4°C下活跃，将表皮除颤体与拉米娜卢西达分离，并将表皮与真皮在1~3小时4、8、10内分离。本报告的目标是优化使用热解质将发炎的老鼠表皮与真皮分离，以检测炎症调解人和神经营养因子的mRNA和蛋白质水平。已提出若干初步报告，包括11、12、13、14、15。这份手稿的目的是描述使用热解质的最佳皮肤分离技术，并证明检测1）炎症标记，2）间柳金-6（IL-6）mRNA，和3）神经生长因子（NGF）mRNA和蛋白质在大鼠表皮与卡拉吉南诱发炎症（C-II）16，17。使用完整的Freund的辅助模型的初步报告显示，NGF mRNA和蛋白质水平在炎症15期间提前增加。在小鼠中，皮肤敏感与牛酮的局部应用导致IL-6 mRNA使用原位杂交36的早期上升。IL-6和NGF都与C-II18、19有牵连，但是没有报告描述IL-6或NGF的mRNA或蛋白质水平，特别是从C-II急性期的表皮。
热解素技术价格低廉，性能简单明了。此外，在炎症过程中，表皮与真皮的热解质分离允许mRNA、西式污点和炎症介导器和神经营养因子的免疫史化学分析。研究者应该能够在皮肤炎症的临床前和临床研究中轻松使用这种技术。
Protocol
该协议遵循俄克拉荷马州立大学健康科学中心（#2016-03）的动物护理指南。 1. 卡拉吉南诱发炎症 （C-II） 麻醉雄性和/或雌性斯普拉格道利大鼠（200×250克;8×9周大）与异黄酮（或注射麻醉剂）。 触摸角膜并轻轻捏左后爪，检查麻醉深度。当动物被适当麻醉时，不会观察到角膜或爪子的反应。 皮下注射正确的玻璃，后爪与100微升的1%（w/v）+卡拉吉南稀释在?…
Representative Results
卡拉吉南注射到大鼠后爪引起典型的炎症症状，如发红和水肿16，17。后爪肿胀是用机械卡钳20测量的。在卡拉吉南治疗之前，每只大鼠的爪子厚度基线值得到，并在6小时和12小时再次测量。与基线值（图1）相比，爪厚显著增加。 鼠光后爪皮的热利辛孵化产生表皮。布莱特菲尔德显微镜用于?…
Discussion
研究确定，大鼠后爪光泽皮肤的表皮很容易与真皮分离，使用热解质（0.5 mG/mL）在PBS与1mM氯化钙在4°C为2.5小时。表皮学评估表明，表皮与地下室膜上的真皮分离，表皮山脊完好无损。热蛋白是由Gram阳性（地理）热蛋白酶24产生的细胞外金属蛋白酶。其活动稳定在4°C，但在10，24，25的宽范围温度?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究的资金由国家卫生研究院NIH-AR047410（KEM）提供
Materials
| λ-carrageenan | Millipore Sigma | 22049 | Subcutaneous injection of carrageenan induces inflammation |
| 7500 Fast Real-Time PCR System | Thermo Fisher Scientific | 4351107 | For RT-PCR analysis |
| Calcium chloride (CaCl2), anhydrous | Millipore Sigma | 499609 | Prevents autolysis of thermolysin |
| Crystal Mount Aqueous Mounting Medium | Millipore Sigma | C0612 | Aqueous mounting medium after toluidine blue staining |
| Donkey anti-Mouse Alexa Fluor 555 | Thermo Fisher Scientific | A-31570 | Secondary antibody for immunohistochemistry |
| Donkey anti-Rabbit IgG, Alexa Fluor 488 | Thermo Fisher Scientific | A-21206 | Secondary antibody for immunohistochemistry |
| Dulbecco's Modified Eagle Medium | Thermo Fisher Scientific | 11966-025 | To maintain tissue integrity |
| Ethylenediaminetetraacetic acid | Millipore Sigma | E6758 | Stops thermolysin reaction |
| Moloney Murine Leukemia Virus (M-MLV) Reverse transcriptase | Promega | M1701 | For complementary DNA synthesis |
| Mouse anti-NGF Antibody (E-12) | Santa Cruz Biotechnology | sc-365944 | For neurotrophin immunohistochemistry |
| ProLong Gold Antifade Mountant | Thermo Fisher Scientific | P36930 | To retard immunofluorescence quenching |
| Rabbit anti-PGP 9.5 | Cedarlane Labs | CL7756AP | For intraepidermal nerve staining |
| SAS Sprague Dawley Rat | Charles River | Strain Code 400 | Animal used for inflammation studies |
| Shandon M-1 Embedding Matrix | Thermo Fisher Scientific | 1310TS | Tissue embedding matrix for tinctorial- and immuno-histochemistry |
| SimpliAmp Thermal Cycler | Thermo Fisher Scientific | A24811 | For RT-PCR analysis |
| SYBR Select Master Mix | Thermo Fisher Scientific | 4472908 | For RT-PCR analysis |
| Thermolysin | Millipore Sigma | T7902 | From Geobacillus stearothermophilus |
| Toluidine Blue | Millipore Sigma | 89640 | For tinctorial staining for brightfield microscopy |
| TRIzol Reagent | Thermo Fisher Scientific | 15596026 | For total RNA extraction for RTPCR |
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Cite This Article
Gujar, V., Anderson, M. B., Miller, K. E., Pande, R. D., Nawani, P., Das, S. Separation of Rat Epidermis and Dermis with Thermolysin to Detect Site-Specific Inflammatory mRNA and Protein. J. Vis. Exp. (175), e59708, doi:10.3791/59708 (2021).