人子宫内膜基质细胞的体外分离蜕膜化
Summary
本研究为人子宫内膜间质细胞的分离和培养进行体外蜕膜化试验提供了一个验证和优化的程序。此外, 本研究还提供了一种利用 siRNAs 在人子宫内膜基质细胞中有效地击倒特定基因的方法。
Abstract
人子宫内膜间质细胞 (人类胚胎干细胞) 从成纤维细胞样的形态分化为分泌性的蜕片, 是胚胎植入子宫内衬的必要转变。不当蜕膜化是导致植入失败和随后的早期胚胎流产的根本原因。因此, 了解蜕膜化的分子机制有利于提高生育成功率。在基于活体的人工蜕膜化的研究中, 往往由于与人类研究相关的伦理难题以及动物模型中的平移并发症而受到限制。因此, 通过原代细胞培养的体外检测, 经常被用来探索通过激素调节蜕膜化的作用。本研究提供了一个详细的协议, 以分离人类胚胎干细胞和随后的人工蜕膜化通过补充激素的培养培养基。进一步, 本研究提供了一个精心设计的方法来击倒任何感兴趣的基因, 利用脂质基 siRNA transfections。该协议允许优化的文化纯度和产品的产量, 从而最大限度地利用这个模型作为一个可靠的方法, 以了解分子机制的基础蜕膜化, 并随后量化decidualized 子宫内膜基质细胞分泌的药物。
Introduction
在月经初潮和更年期的阶段之间, 育龄妇女每月进行激素调节的子宫内膜增生、分化以及随后脱落的妊娠准备过程, 这一进程被称为1。 ,2。人类子宫内膜的这种物理修饰是必要的适当的胚胎植入到子宫壁1。子宫内膜的改变, 包括形态学和生物化学的适应, 通过卵巢类固醇激素雌激素和孕激素 (P4)3,4,5在整个月经周期中进行调解。在增生 (或卵泡) 阶段, 小鼠雌激素水平增加, 引发子宫内膜增厚。排卵后, 分泌 (或黄体) 阶段促进 P4 浓度的显著上升, 诱导子宫内膜基质细胞 (ESC) 从成纤维细胞样的外观到圆形, 上皮样蜕细胞的形态学改变。在一个被称为蜕膜化4,6的过程中。不正确的蜕膜化是导致植入失败和随后的早期胚胎流产4,7,8的根本原因。因此, 了解蜕膜化的分子机制有利于早期妊娠丢失的诊断和治疗。
目前, 利用几种方法探讨蜕膜化对子宫内膜基质细胞的潜在影响。在体内, 小鼠子宫可以通过机械刺激 (即, 划伤) 或注油在荷尔蒙引子宫9中人工诱导蜕膜化。与人类不同的是, 这种合成刺激通过提供囊胚存在的外观促进子宫腔的分化, 这是在啮齿目动物10,11中启动蜕膜化所需的步骤。因此, 由于与动物模型相关的翻译并发症和人类体内研究中的伦理困境, 蜕膜化模型是最成功的体外研究方法。
在这项研究中, 通过在当地的英语和西班牙报纸上放置广告来招聘科目。被确定为这项研究的合适人选的科目被引入与研究协调员会面, 其中讨论了潜在风险的充分披露。在确认对所涉潜在风险的完全理解后, 受试者的同意以书面和口头形式获得。主题同意包括准许 (1) 接受放血 (2) 长期保存他们的组织为将来研究目的和 (3) 同意创造原始的文化从被收集的组织标本。经同意后, 受试者获得一个表格, 以完成允许自我识别种族/族裔和/或保密权利。随后的访问将根据主题的月经周期来实现子宫内膜活检。被招募参加这项研究的志愿者反映了2012人口普查所记录的圣路易斯大都市地区的种族和种族人口, 不涉及任何弱势群体, 包括孕妇、胎儿、胚胎、18岁以下儿童或其他弱势群体。参加活检标本收集的资格要求包括 (1) 年龄在18-45 岁 (2) 之间, 有定期月经周期 (25-32 天) (3), 没有当前怀孕或使用荷尔蒙/宫内节育器避孕药具在注册前30天 (4) 没有当前的阴道感染或性传播疾病 (5) 没有目前的抗生素治疗, (6) 没有当前异常的 Pap 涂片。
在这项研究中, 人类子宫内膜基质细胞 (人类胚胎干细胞) 被培养和人工诱导通过补充激素 (雌二醇 (E2), 醋酸甲羟孕酮 (MPA) 和环腺苷来进行体外蜕膜化单磷酸 (营)) 到培养基中。在这种方法中, 蜕膜化的程度是根据激素治疗的总天数而改变的。与骨架重排, 激素补充诱导的生物化学适应, 其中蜕细胞经历分泌样素质2,4。标记基因的表达, 如催乳素和胰岛素样生长因子结合蛋白 1 (IGFBP1), 可以用来确认和量化人类胚胎干细胞蜕膜化的程度5,12,13,14. 重要的是, 这项议定书是否有能力进行基因特定的击倒, 也证明了这一点。
Protocol
Representative Results
Discussion
女性生殖月经周期的特点是孕激素水平在整个黄体期的上升, 从而诱导蜕膜化到圆形, 上皮样分泌细胞3,8。蜕膜化的启动是物种依赖性的。在人类中, 蜕膜化自发发生在孕激素浓度上升, 而小鼠需要囊胚存在10,11。这种不一致的蜕膜化例证了研究细胞分化的人细胞系的转化优势。通过原代细胞培养的体外检测, 经…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者没有什么可透露的。
Materials
| Opti-MEM I (1X) Reduced Serum Medium | Gibco | 31985-070 | For decidualization media |
| DMEM / F12 (1:1) (1X) | Gibco | 11330-032 | |
| Trypan Blue Stain (0.4%) | Gibco | 15250-061 | For cell count |
| PureLink RNA Mini Kit | Invitrogen | 12183018A | For RNA Isolation/Purification |
| TaqMan 2X Universal PCR Master Mix | Applied Biosystems | 4304437 | |
| High Capacity cDNA Reverse Transcription Kit | Applied Biosystems | 4374967 | |
| Eukaryotic 18S rRNA Endogenous Control | Life Technologies | 4319413E | For qPCR internal control |
| TaqMan Gene Expression Assay Prolactin Probe | Applied Biosystems | 4331182 | For qPCR internal control |
| TaqMan Gene Expression Assay IGFBP1 Probe | Applied Biosystems | 4331182 | For qPCR internal control |
| Phalloidin-iFluor 488 Reagent – CytoPainter | Abcam | ab176753 | |
| Human Prolactin ELISA Kit | Invitrogen | EHIAPRL | |
| 16% Paraformaldehyde | Alfa Aesar | 30525-89-4 | Fixative |
| Lipofectamine RNAiMAX | Invitrogen | 13778150 | Transfection Reagent |
| ProLong Gold antifade reagent with DAPI | Invitrogen | P36935 | For mounting |
| 0.25% Trypsin-EDTA (1X) | Gibco | 25200-056 | |
| Penicillin Streptomycin | Gibco | 15140-122 | |
| Charcoal Stripped Fetal Bovine Serum | Sigma | F6765-500ML | |
| Sodium Bicarbonate (7.5%) | Gibco | 25080-094 | |
| Ficoll-Paque PLUS Reagent | Fisher Scientific | 45001749 | |
| Collagenase from Clostridium histolyticum | Sigma | C0130-1G | |
| Deoxyribonuclease I from bovine pancreas | Sigma | DN25-100MG | |
| Medroxyprogesterone 17-acetate | Sigma | M1629-1G | For EPC Media |
| Estradiol | Sigma | E1024-1G | For EPC Media |
| cAMP | Sigma | A6885-100MG | For EPC Media |
| Fine straight stitch scissors | Fine Science Tools | 15396-00 | |
| TaqMan Gene Expression Assay NCOA2 Probe | Applied Biosystems | 4351372 | |
| Fetal Bovine Serum, heat inactivated | Gibco | 10-082-147 | |
| Antibiotic-antimycotic | Thermo Scientific | 15240062 | |
| Hank’s Balanced Salt Solution | Corning | 21-021-cv | |
| 50 mL conical polypropylene Falcon tube | Corning | 352098 | |
| Dumont #5/45 Forceps | Fine Science Tools | 11251-35 | |
| 100 x 15 mm Glass Petri dish | VWR | 75845-546 | |
| 40 micron cell strainer | Midsci | 229481 | |
| Isotemp 215 Water bath | Fisher Scientific | FS-215 | |
| LSE Centrifuge | Corning | 6755 | |
| Human NCOA2 siRNA | Dharmacon | L-020159-00 | Gene specific qPCR probe |
| Steri-cycle i160 CO2 Incubator | Thermo Scientific | 51030301 | |
| NanoDrop 2000 | Thermo Scientific | ND-2000 | For RNA quantification |
| Phosphate Buffered Saline | Fisher Scientific | 50146771 | |
| 75 cm Canted Neck Cell Culture flask | Corning | 430641U | |
| 6 well Non-Pyrogenic Cell Culture Plate | Corning | 3506 | |
| Pipet Controller Ultra | Corning | 4099 | |
| Photoshop | Adobe | 19.0.1.334 | |
| 25 cm Canted Neck Cell Culture flask | Corning | 7200876 | |
| Triton X-100 | Sigma | X100-1L | |
| 15 mL conical polypropylene Falcon tube | Corning | 352099 | |
| 10 mL Syringe | BD | 302995 | |
| 1300 Series A2 Biological Safety Hood | Thermo Scientific | 1377 | |
| accuspin Micro17 centrifuge | Fisher Scientific | 13100675 | |
| 7500 Fast real time PCR system | Applied Biosystems | 3052632 | |
| EVOS FL Immunofluorescence Microscope | Life Technologies | 01414-155G-291 | |
| Leica Inverted Light Microscope | Leica | DMi1 | |
| Illrustrator | Adobe | 22.0.1.253 | |
| Excel Spreadsheets | Microsoft | 2016 | |
| Deckglaser 18 mm cover glasses | NeuVitro | GG-18 | |
| Reichert Bright-Line Hemacytometer | Sigma | Z359629-1EA | |
| 2-mercaptoethanol | Fisher Bioreagents | BP176-100 | For RNA lysis buffer |
| 1.2mL External Threaded Polypropylene Cryogenic Vial | Corning | 430658 | For freezing HESC cells |
| Dimethyl Sulfoxide (DMSO) | Sigma | D2650-100mL | For freezing media |
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