从肉鸡雏鸡胚胎中分离出前脂肪细胞
Summary
本方案描述了一种从肉鸡胚胎中的脂肪组织中分离前脂肪细胞的简单方法。该方法能够高产率、原代培养和前脂肪细胞的脂肪分化进行分离。油红O染色和脂质/DNA染色测量了分化培养基诱导的分离细胞的脂肪生成能力。
Abstract
原代前脂肪细胞是了解控制脂肪细胞分化和代谢的分子途径的有价值的实验系统。鸡胚胎提供了从脂肪发育的最早阶段分离前脂肪细胞的机会。这种原代细胞可用于鉴定影响前脂肪细胞增殖和脂肪分化的因素，使其成为与儿童肥胖和控制家禽中多余脂肪沉积相关的研究的宝贵模型。产后脂肪组织的快速生长通过将其从肉鸡的肌肉生长中分配出来有效地浪费饲料。因此，了解脂肪组织发育最早阶段的方法可能为调节这种趋势提供线索，并确定在生命早期限制脂肪扩张的方法。本研究旨在开发一种有效的方法，用于从商业肉鸡（肉型）雏鸡胚胎的发育脂肪组织中分离出的前脂肪细胞的分离，初级培养和脂肪分化。该过程已经过优化，以产生具有高活力（约98%）的细胞，并增加了分化成成熟脂肪细胞的能力。这种简单的胚胎前脂肪细胞分离、培养和分化方法支持早期脂肪生长和发育的功能分析。
Introduction
肥胖对成人和儿童都是一种全球健康威胁。超重或肥胖的儿童肥胖的可能性大约是成年的五倍，使他们患心血管疾病、糖尿病和许多其他合并症的风险显著增加。大约13.4%的美国2-5岁儿童患有肥胖症1，这表明积累多余身体脂肪的倾向可以在生命早期启动。由于非常不同的原因，过量脂肪组织的积累是肉鸡（肉型）鸡关注的问题。现代肉鸡非常高效，但仍然积累的脂质比生理上必需的多2，3.这种趋势在孵化后不久就开始了，并通过将其从肌肉生长中分配出来，有效地浪费了饲料，这是最昂贵的生产成分。因此，对于儿童和肉鸡，尽管出于非常不同的原因，但需要了解影响脂肪组织发育的因素，并确定在生命早期限制脂肪扩张的方法。
脂肪细胞形成于前脂肪细胞，脂肪组织来源的干细胞，经过分化以发展成熟的脂质储存脂肪细胞。因此， 体外 前脂肪细胞是肥胖研究的宝贵实验模型。这些从脂肪库的基质血管部分分离出来的细胞，可以提供对控制脂肪细胞分化和新陈代谢的分子途径的基本理解4，5。雏鸡胚胎是发育研究中一种有利的实验模型，因为按照所需的时间表培养卵子使实验操作更容易，因为它可以在没有母亲牺牲的情况下获得胚胎，以观察胚胎的一系列发育阶段。此外，相对于较大的动物模型，不需要复杂的外科手术和漫长的时间来获得胚胎。因此，雏鸡胚胎提供了从脂肪组织发育的最早阶段获得前脂肪细胞的机会。皮下脂肪组织在胚胎第12天（E12）左右的雏鸡中变得可见，作为位于大腿周围的明确定义的库。该库富含高度增殖的前脂肪细胞，这些前脂肪细胞在发育线索下积极经历分化以形成成熟的脂肪细胞6，7。脂肪分化的过程在鸡和人类之间是可比的。因此，从雏鸡胚胎中分离出的前脂肪细胞可以作为与人类和家禽相关的研究的两用模型。然而，随着细胞生长成成熟的脂肪细胞，前脂肪细胞的产量随着年龄的增长而下降5。
本方案优化了在肉鸡雏鸡胚胎中脂肪细胞分化和脂肪细胞肥大处于峰值的阶段（E16-E18）期间从脂肪组织中分离前脂肪细胞8。该过程可以评估发育 中的胚胎在卵中暴露的因素（例如母鸡饮食）对脂肪细胞发育和 离体脂肪电位的影响。它还可以测试各种操作（例如，缺氧、营养物质添加、药理激动剂和拮抗剂）对脂肪细胞祖细胞的脂肪生成或各种’组，代谢组，甲基组）的影响。作为脂肪形成最早阶段的表示，使用该方案获得的细胞是与家禽和人类相关的研究的宝贵模型。
Protocol
所有动物程序均由田纳西大学机构动物护理和使用委员会批准。新鲜受精的商业肉鸡蛋（Cobb 500）是从当地的孵化场获得的。将卵在38°C下以60%的相对湿度孵育，直到胚胎第16-18天解剖（E16-E18）。从皮下（股骨）库收集脂肪组织。 1. 隔离和培养的准备 准备培养罩和仪器。 在开始解剖之前，请在层流罩中设置一个工作区。通过用70%乙醇拭子对工作区域…
Representative Results
原代前脂肪细胞在形态上类似于成纤维细胞，具有不规则的星形形状和中央核（图2A-C）。细胞容易粘附在组织培养塑料上，并在附着后不久开始增殖。当在培养基中提供脂肪酸时，它们迅速分化并积累脂质液滴（图3D）。此处代表的分离物中报告的生存能力（98%，基于染料排除）是典型的。虽然细胞相当健壮，但在分离过程中…
Discussion
尽管一些描述良好的方案已经报道了前脂肪细胞14，15，16，17的分离，但胚胎前脂肪细胞的分离已经优化，可用于肉鸡雏鸡早期脂肪生长和发育的功能分析。该方案产生具有高分化潜力的高生存能力的胚胎脂肪细胞祖细胞。此外，所提出的分离前脂肪细胞的程序不仅限于胚胎，还可以用于孵化后的雏?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢UT农业研究和动物科学系支持和优化该协议。这项工作由美国农业部赠款资助。
Materials
| 1 mL Pipette | Eppendorf | Z683825 | Single Channel Pipette, 100 – 1000 µL |
| 1 mL Pipette Tip | Fisher Scientific | 02-707-402 | |
| 100% Isopropanol | Fisher Scientific | A426P4 | |
| 1x PBS | Gibco | 10010023 | |
| 25 mL Flask | Pyrex | 4980-25 | |
| 37% Formaldehyde | Fisher Scientific | F75P-1GAL | |
| 6-Well Plate | Falcon | 353046 | Tissue Culture-treated |
| 96-Well Assay Plate | Costar | 3632 | |
| 96-Well Plate, Black Bottom | Costar | 3603 | Tissue Culture-treated |
| AdipoRed | Lonza | PT-7009 | |
| Amphotericin B | Gibco | 15290026 | |
| Bench Top Wiper (Kimtechwiper) | Kimberly-Clark | 34155 | |
| Betadine | Up & Up | NDC 1167300334 | 20% Working Solution |
| Cell Counter | Corning | 6749 | |
| Cell Strainer, 40 µm | SPL | 93040 | |
| Centrifugaton | Eppendorf | 5702 | |
| Chicken Serum | Gibco | 16110082 | |
| Conical Centrifuge Tubes, 15 mL | VWR | 10025-690 | |
| Conical Centrifuge Tubes, 50 mL | Falcon | 352098 | |
| Cryovial | Nunc | 343958 | |
| Curved Forceps, 100 mm | Roboz Surgical | RS-5137 | |
| Curved Surgical Scissors, 115 mm | Roboz Surgical | RS-6839 | |
| Distilled Water | Millipore | SYNSV0000 | Despensed as needed |
| DMEM/F12 | HyClone | SH30023.01 | |
| DMSO | Sigma | D2650 | |
| Ethanol | Decon Labs | 2701 | 70% Working Solution |
| Fetal Bovine Serum (FBS) | Gibco | 10437028 | |
| Fluorescent Microscope | Evos | M7000 | |
| Fluorescent Plate Reader | Biotek | Synergy H1 | |
| Foil | Reynolds | Reynolds Wrap Heavy Duty Aluminum Foil, 125 SQ. FT. | |
| Freezing Container | Thermo Scientific | 5100-0001 | |
| Gelatin | Millipore | 4055 | 2% Working Solution |
| Hematocytometer (Counting Chamber) | Corning | 480200 | 0.1 mm deep |
| Incubator | Fisher Scientific | 6845 | |
| Instrument Sterilizer | VWR | B1205 | |
| Linoleic Acid-Oleic Acid-Albumin | Sigma | L9655 | 1x Working Solution |
| Microscope | Evos | AMEX1000 | |
| Multi-Channel Pipette | Thermo Scientific | 4661070 | 12-Channel Pipetters, 30 – 300 µL |
| Na2HPO4 | Sigma | S-7907 | |
| NaH2PO4 | Sigma | S-3139 | |
| NucBlue | Invitrogen | R37605 | |
| Oil Red O | Sigma | O-0625 | |
| Orbital Shaker | IKA | KS130BS1 | |
| Paper Towel | Tork | RK8002 | |
| Parafilm | Parafilm M | PM996 | |
| Penicillin/Steptomycin (P/S) | Gibco | 15140122 | 1x Working Solution |
| Petri dishes, 100 mm | Falcon | 351029 | |
| Petri dishes, 60 mm | Falcon | 351007 | |
| Plate Shaker | VWR | 200 | |
| RBC Lysis Buffer | Roche | 11814389001 | |
| Reagent Reservior | VWR | 89094-680 | |
| Small Beaker, 100 mL | Pyrex | 1000-100 | |
| Spectrophotometer Plate Reader | Biotek | Synergy H1 | |
| Sterile Gauze | McKesson | 762703 | |
| Straight Forceps, 120 mm | Roboz Surgical | RS-4960 | |
| Straight Scissors, 140 mm | Roboz Surgical | RS-6762 | |
| T-25 Flask | Corning | 430639 | Tissue Culture-treated |
| Tissue Culture Incubator | Thermo Scientific | 50144906 | |
| Tissue Strainer, 250 µm | Pierce | 87791 | |
| Trypan Blue Stain | Gibco | 15250061 | |
| Trypsin | Gibco | 15400054 | 0.1% Working Solution |
| Tweezers, 110 mm | Roboz Surgical | RS-5035 | |
| Type 1 Collagenase | Gibco | 17100017 | |
| Water Bath | Fisher Scientific | 15-462-10 | |
| Whatman Grade 1 Filter Paper | Whatman | 1001-110 |
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Cite This Article
Kim, M., Jung, U., Shepherd, E., Mihelic, R., Voy, B. H. Isolation of Preadipocytes from Broiler Chick Embryos. J. Vis. Exp. (186), e63861, doi:10.3791/63861 (2022).