人体滋养层模型研究母体肥胖炎症对胎盘自噬的调节作用
Summary
本文介绍了一种用于人胎盘绒毛组织的取样方法, 其次是分离细胞原代细胞培养。滋养治疗 TNFα重述炎症在肥胖的宫内环境和促进发现的分子靶调控的炎症在胎盘孕产妇肥胖。
Abstract
孕产妇肥胖与不良围产期结局的风险增加有关, 可能是由胎盘功能受损引起的, 部分原因是自噬的失调。肥胖妊娠胎盘中自噬调节因子表达的异常变化可能由与肥胖和妊娠有关的炎症过程调节。这里描述的是一个协议的绒毛组织和绒毛细胞的分离, 从一项人类胎盘的原代细胞培养。其次是一种方法模拟的炎症环境, 在肥胖的宫内环境中, 治疗原发性滋养从精妊娠与肿瘤坏死因子α (TNFα), 一个促炎细胞因子, 是升高的肥胖和怀孕.通过这里所描述的协议的实施, 发现暴露于外源性 TNFα调节了滋养的表达, 这是一种自噬的负调节, 来自于女性胎儿的精益妊娠。虽然在肥胖的宫内环境中的各种生物因素保持了在滋养中调节关键通路的潜能, 这个ex 体内系统特别适用于确定表达模式是否观察到了体内在人类胎盘中, 孕产妇肥胖是 TNFα信号的直接结果。最终, 这种方法提供了一个机会来分析与母体肥胖有关的炎症的调控和分子影响, 对滋养的自噬和其他重要的细胞通路有可能影响胎盘功能。
Introduction
肥胖是一种炎症状态, 其特点是慢性低度炎症, 来源于过量的脂肪组织和养分的供应。在肥胖症中, 促炎细胞因子在代谢组织中升高, 并在循环系统中具有系统性。一个强有力的证据显示, TNFα在肥胖的设置中显著升高, 而胰岛素抵抗和代谢功能障碍的影响是1。活化 TNFα也有助于疾病的发病机制, 如癌症和自身免疫, 使其成为一个有吸引力的治疗目标2。
肥胖的炎症是由妊娠加重的, 也是一个炎症状态3,4。以前的结果表明, 胎盘 TNFα的含量随着孕产妇的肥胖而增加。此外, TNFα治疗抑制雌性而非雄性滋养层细胞的线粒体呼吸, 表明 TNFα参与了以性二方式调节胎盘代谢的作用5。孕产妇肥胖与妊娠期间各种并发症的发病率增加有关, 包括死胎, 男性胎儿最易受感染3,6,7,8.由于其在母体-胎儿界面的关键作用, 在肥胖宫内环境中胎盘功能的变化对炎症信号的反应可能在调节肥胖妊娠结局中起重要作用。
细胞和合体在胎盘的绒毛状组织是关键的内分泌信号和营养和氧气交换之间的母亲和发育的胎儿9。绒毛细胞 (以下简称滋养) 功能能力的紊乱可能危及胎儿的健康和发育。本协议描述了一种从人类足月胎盘中提取绒毛组织的方法, 通过剥离绒毛膜和基底板, 以及一个优化的分离滋养原细胞培养的程序。该协议是从建立的方法, 涉及酶消化的绒毛组织, 释放细胞从胞外基质后, 差密度离心分离滋养10, 11,12。本议定书详细介绍了一种方法, 其中主要滋养来自胎盘从精益怀孕的治疗与培养基辅以 TNFα, 以模拟一个组成部分的炎症环境与孕产妇肥胖。最后, 本文介绍了一种从 TNFα处理后的滋养中提取总细胞裂解的方法, 并对其进行了探讨, 以检测基因表达的变化。
虽然此模型不重述致在子宫内环境中的整体, 但它提供了一个受控系统, 允许您解析 TNFα介导的炎症在滋养的反应中的个人贡献孕妇肥胖症这个模型既提供了发现或确认分子靶点的机会直接调控的 TNFα信号在滋养, 并且允许一个测试, 如果基因表达模式的变化观察到的在体内在胎盘与母体肥胖可能是 TNFα介导的炎症的结果。
本文所介绍的方法是为了测试 TNFα介导的炎症对人类滋养自噬的调节作用。滋养从肥胖怀孕与男性胎儿陈列被打乱的噬转交, 或者 autophagosome 成熟13。一种称为卢比的蛋白质 (运行域蛋白 Beclin1-interacting 和富含半胱氨酸的含), 这是本地化的溶酶体和晚期体, 最近被描述为 “刹车” 在噬的周转过程中, 因为它的功能作为一个负autophagosome 成熟的调节器14,15。事实上, 卢比是抑制自噬的一个罕见的例子, 它使它成为一个有价值的治疗目标。很少有关于卢比的病理生理学意义的信息, 除了它在先天免疫应答微生物16、17和心肌细胞保护18中的作用。使用这里描述的协议, 它发现, 卢比是上调在女性原发性滋养的反应, 以增加浓度的 TNFα高达 250 pg/毫升的治疗。在孕产妇肥胖时, 对卢比的调节可能会影响女性胎儿的体重。综述炎症与孕产妇肥胖相关前体通过暴露人滋养外源性 TNFα提供了一个平台, 研究肥胖宫内环境对关键通路调节的影响滋养和延伸, 胎盘功能。
Protocol
Representative Results
Discussion
胎盘, 负责调节胎儿的生长, 在肥胖环境中表现出损害性功能6。尽管滋养的代谢要求很高, 但母体肥胖的胎盘表现出功能失调的线粒体呼吸6,19。胎盘代谢的变化可能有助于增加并发症的发病率和不良胎儿结局观察到肥胖怀孕3,6,7。在母体肥胖的胎盘中, 自噬也会受到?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢那些为这项研究捐出胎盘的妇女。我们还感谢站和产妇和胎儿研究小组协调收集胎盘的劳动和分娩部。我们感谢埃里克. 王博士和凯利, 他对实验方法和优化的支持和帮助。
这项工作由 NIH HD076259A (am) 和阿哈 GRNT29960007 (am) 资助。
Materials
| 10X HBSS | Gibco | 14185-052 | |
| CaCl2 (anhyd.) | Sigma-Aldrich | C1016-100G | |
| MgSO4 (anhyd.) | Sigma-Aldrich | M7506-500G | |
| Hepes | Fisher Scientific | BP310-500 | |
| Trypsin | Gibco | 15090-046 | |
| DNAse | Worthington Biochemical Corp. | LS002139 | |
| Protease/Phosphatase inhibitors | Thermofisher Scientific | 88668 | |
| Tris HCl | Invitrogen | 15506-017 | |
| EDTA | Invitrogen | 15576-028 | |
| NaCl | Sigma-Aldrich | S7653-1KG | |
| SDS | Fisher Scientific | BP166-600 | |
| Sodium deoxycholate. | Fisher Scientific | AAJ6228822 | |
| Triton X-100 | Sigma-Aldrich | X100-500ML | |
| Iscove’s Modified Dulbecco’s Medium (IMDM) | Gibco | 12440-046 | |
| Fetal Bovine Serum (FBS) | Corning | 35-010-CV | |
| Neonatal Calf Serum (NCS) | Gibco | 26010-074 | |
| Penicillin/Streptomycin (Pen/Strep) | Gibco | 15140-122 | |
| 10% Formalin | Fisher Scientific | 23-427-098 | |
| DMSO | Sigma-Aldrich | D2650-100ML | |
| TNFα | Sigma-Aldrich | SRP3177-50UG | |
| Phosphate Buffered Saline (PBS) | Gibco | 70013-032 | |
| K2EDTA vacutainer blood collection tubes | BD | 366450 | |
| Percoll (Density Gradient Media, DGM) | GE Healthcare | 17-0891-01 | |
| 6 well plates | Corning | 353046 | |
| Cell strainers | Fisher Scientific | 22363549 | |
| Eppendorf Safe-Lock Tubes 2.0 mL, natural | Fisher Scientific | 22363352 | |
| Trypan Blue | Corning | 25-900-Cl | |
| Bio-Rad Mini-PROTEAN Tetra System | Bio-Rad | 1658001FC | |
| Bio-Rad Mini Trans-Blot Cell | Bio-Rad | 1658033 | |
| TGX FastCast Acrylamide Kit, 12% | Bio-Rad | 1610175 | |
| Mini-Protean 3 Multi-Casting Chamber | Bio-Rad | 1654112 | |
| 4X Laemmli Sample Buffer | Bio-Rad | 1610747 | |
| 2-Mercaptoethanol | Sigma-Aldrich | M3148-100ML | |
| Glycine | Bio-Rad | 1610718 | |
| Tween-20 | Sigma-Aldrich | P7949-500ML | |
| Instant Nonfat Dry Milk | Carnation | ||
| Rubicon (D9F7) Rabbit mAb | Cell Signalling Technology | 8465S | |
| Monoclonal Anti-β-Actin antibody produced in mouse | Sigma-Aldrich | A2228-100UL | |
| Anti-rabbit IgG, HRP-linked Antibody | Cell Signalling Technology | 7074S | |
| Anti-mouse IgG, HRP-linked Antibody | Cell Signalling Technology | 7076S | |
| SuperSignal West Pico PLUS Chemiluminescent Substrate | Thermo Scientific | 34578 |
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