人类羊膜不同解剖区域的皮下细胞体外培养
1Departamento de Fisiología y Desarrollo Celular,Instituto Nacional de Perinatología, 2Biotecnología Médica y Farmacéutica CONACYT,Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ), 3Departamento de Inmunobioquímica,Instituto Nacional de Perinatología, 4Instituto de Neurobiología, UNAM
Summary
该协议描述了从人类羊膜的不同解剖区域分离上皮细胞，以确定其异质性和功能特性，以便可能应用于临床和物理病理学模型。
Abstract
文献中报告了关于人类羊皮上皮细胞（HAEC）的隔离和培养的若干协议。然而，这些假设羊皮上皮是同质层。人类羊膜可分为三个解剖区域：反射、胎盘和脐带。每个区域有不同的生理作用，如在病理条件下。在这里，我们描述了一个协议，解剖人类羊羊组织在三个部分，并在体外维护它。在培养中，从反射羊膜中提取的细胞呈长方体形态，而来自胎盘和脐带的细胞是鳞状的。尽管如此，所有获得的细胞都有上皮表型，通过E-cadherin的免疫检测证明。因此，由于原位胎盘和反射区域在细胞成分和分子功能上有所不同，因此体外研究可能有必要考虑这些差异，因为它们可能对HAEC的使用产生生理影响。生物医学研究及这些细胞在再生医学中的可喜应用。
Introduction
人类羊皮上皮细胞（HAEC）起源于胚胎发育的早期阶段，在受精后8天左右。它们产生于从羊膜1的最内层衍生的表征细胞的鳞状上皮细胞。因此，HAEC被认为是表征中多能细胞的残余物，有可能分化成胚胎2的三个生殖层。在过去的十年中，不同的研究小组已经开发出了在妊娠期内将这些细胞从羊膜分离出来的方法，以在体外3、4的培养模型中描述其推定多能相关特性。
因此，已发现HAEC具有人类多能干细胞（HPSC）的特征特征，如表面抗原SSEA-3、SSEA-4、TRA 1-60、TRA 1-81;多能转录因子OCT4、SOX2和NANOG的核心;和扩散标记KI67，表明他们是自我更新5，6，7。此外，这些细胞已经受到挑战，使用分化协议获得细胞阳性的生殖层（外生，中生代和内皮）4，5，8，以及人类疾病的动物模型。最后，HAEC表示E-cadherin，这表明他们保留了上皮性质很像HPSC5，9。
除了胚胎来源外，HAEC还有其他内在特性，使它们适合不同的临床应用，如抗炎和抗菌分子的分泌10，11，生长因子和细胞因子释放12，不形成畸马细胞瘤时，他们移植到免疫缺陷小鼠与HPSC2对比，和免疫耐受性，因为它们表达HLA-G，减少排斥后的风险移植13.
然而，以前的报告假设人类羊膜是同质膜，没有考虑到它可以在解剖学和生理上分为三个区域：胎盘（覆盖去皮底的羊膜），脐带（包围脐带的部分），并反射（膜的其余部分不附着在胎盘上）14 。研究表明，羊驼的胎盘和反射区域在形态学、线粒体活性、活性氧物种15检测、miRNA表达16和信号通路17激活方面存在差异。这些结果表明，人类羊皮被一个具有不同功能的异质人群所整合，在原位或体外模型中进行进一步的研究应考虑。虽然其他实验室已经设计了从整个膜分离HAEC的协议，但我们的实验室已经建立了一个协议来分离、培养和表征来自不同解剖区域的细胞。
Protocol
该协议由墨西哥城国家研究所伦理委员会批准（注册号212250-21041）。这些研究中进行的所有程序均符合《国家佩里纳洛尼亚研究所》、《赫尔辛基宣言》和卫生部《墨西哥官方标准》中规定的指导方针的道德标准。 1. 准备 使用 EDTA 准备 1x PBS 解决方案。为此，将 500 μL 的 0.5 M EDTA 库存添加到 500 mL 的 1x PBS 中，最终浓度为 0.5 mM EDTA。 为 HAEC 准备培养基。服用450 …
Representative Results
HAEC从羊膜的三个解剖区域分离出来，并在体外单独培养。在培养48小时后，具有上皮表型的细胞附着在板表面，尽管介质中也含有细胞碎片和浮细胞，一旦介质改变，这些细胞被移除（图3）。 在初级培养物（通道零，P0）的处理过程中，可能会出现一些并发症，从而干扰实验数据分析（图4）：在识别试剂污染或分离过程中存在细?…
Discussion
我们实现了一种新的协议，将HAEC与术语膜分离开来。它不同于以前的报告，因为每个膜在分离前被分成三个解剖区域，以分析每个膜的细胞。
该协议中最关键的步骤之一是清洗膜以去除所有血块，因为它们在分离上皮细胞时会干扰胰蛋白酶的活动。未能正确执行此步骤可能导致获得具有过量红细胞和很少粘附上皮细胞的主要培养物。如果在设定细胞种子后的前 24-48 小时未观…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们的研究得到了墨西哥国家研究所（21041年和21081年）和国家委员会（A1-S-8450和252756）的资助。我们感谢杰西卡·冈萨雷斯·诺里斯和莉迪亚·尤里娅·帕里德斯·维维斯的技术支持。
Materials
| Culture reagents | |||
| 2-Mercaptoethanol | Thermo Fisher Scientific/Gibco | 21985023 | 55 mM |
| Animal-Free Recombinant Human EGF | Peprotech | AF-100-15 | |
| Antibiotic-Antimycotic | Thermo Fisher Scientific/Gibco | 15240062 | 100X |
| Dulbecco's Modified Eagle Medium | Thermo Fisher Scientific/Gibco | 12430054 | Supplemented with high glucose and HEPES |
| EDTA | Thermo Fisher Scientific/Ambion | AM9260G | 0.5 M |
| Embryonic stem-cell FBS, qualified | Thermo Fisher Scientific/Gibco | 10439024 | |
| Non-Essential Amino Acids | Thermo Fisher Scientific/Gibco | 11140050 | 100X |
| Paraformaldehyde | any brand | ||
| Phosphate-Buffered Saline | Thermo Fisher Scientific/Gibco | 10010023 | 1X |
| Saline solution (sodium chloride 0.9%) | any brand | ||
| Sodium Pyruvate | Thermo Fisher Scientific/Gibco | 11360070 | 100 mM |
| Trypsin/EDTA 0.05% | Thermo Fisher Scientific/Gibco | 25300054 | |
| Disposable material | |||
| 100 µm Cell Strainer | Corning/Falcon | 352360 | |
| 100 mm TC-Treated Culture Dish | Corning | 430167 | |
| 24-well Clear TC-treated Multiple Well Plates | Corning/Costar | 3526 | |
| 6-well Clear TC-treated Multiple Well Plates | Corning/Costar | 3516 | |
| Non-Pyrogenic Sterile Centrifuge Tube | any brand | with conical bottom | |
| Non-Pyrogenic sterile tips of 1,000 µl, 200 µl and 10 µl. | |||
| Sterile cotton gauzes | |||
| Sterile serological pipettes of 5, 10 and 25 mL | any brand | ||
| Sterile surgical gloves | any brand | ||
| Equipment | |||
| Biological safety cabinet | |||
| Centrifuge | |||
| Micropipettes | |||
| Motorized Pipet Filler/Dispenser | |||
| Sterile beakers of 500 mL | |||
| Sterile plastic cutting board | |||
| Sterile scalpels, scissors, forceps, clamps | |||
| Sterile stainless steel container | |||
| Sterile tray | |||
| Tube Rotator | MaCSmix | ||
| Antibodies and Kits | Antibody ID | ||
| Anti-E-cadherin | BD Biosciences | 610181 | RRID:AB_3975 |
| Anti-KI67 | Santa Cruz | 23900 | RRID:AB_627859) |
| Anti-NANOG | Peprotech | 500-P236 | RRID:AB_1268274 |
| Anti-OCT4 | Abcam | ab19857 | RRID:AB_44517 |
| Anti-SOX2 | Millipore | AB5603 | RRID:AB_2286686 |
| Anti-SSEA-4 | Cell Signaling | 4755 | RRID:AB_1264259 |
| Anti-TRA-1-60 | Cell Signaling | 4746 | RRID:AB_2119059 |
| Goat Anti-Mouse Alexa Fluor 488 | Thermo Fisher Scientific | A-11029 | RRID:AB_2534088 |
| Goat Anti-Rabbit Alexa Fluor 568 | Thermo Fisher Scientific | A-11036 | RRID:AB_10563566 |
| Tunel Assay Kit | Abcam | 66110 |
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Cite This Article
Avila-González, D., García-López, G., Díaz-Martínez, N. E., Flores-Herrera, H., Molina-Hernández, A., Portillo, W., Díaz, N. F. In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane. J. Vis. Exp. (153), e60551, doi:10.3791/60551 (2019).