小鼠尿的解剖和外植体培养对囊附着的作用分析

Summary

我们描述了一个体外测定模型囊附着, 第一步胎盘形成。该协议展示了小鼠 allantoides 在固定化α4β1整合素上的解剖和外植体培养。尿附件是在预定的时间点显微镜下评估的。

Abstract

胎盘对哺乳动物胚胎的生长发育至关重要。由于这个原因, 许多基因的改变和环境的侮辱, 扰乱胎盘的发展或功能可能导致早孕损失的小鼠和人类。然而, 简单的体外检测对胎盘形成的潜在影响是缺乏的。在这里, 我们的重点是建模的第一步和关键步骤的胎盘形成, 其中包括附着的尿的绒毛膜。我们描述了一种快速评估囊外植体附着在固定化α4β1整合素上的方法, 它是一种 chorio 模拟的基底。这种体外方法可以对不同连续时间点的多个尿的附着和扩散行为进行定性评估。该协议可用于调查有针对性的小鼠突变, 药物, 或各种环境因素的影响, 已与妊娠并发症或胎儿损失的尿附着在前体内。

Introduction

胎盘在子宫环境中的胚胎生长发育是必不可少的。它构成了胎儿和母体循环之间的氧气、代谢物和养分交换的界面, 也是内分泌和免疫器官的功能。任何内生或外源性侮辱, 损害胎盘发育可能导致宫内发育迟缓, 胎儿的损失, 或怀孕并发症, 在小鼠和人类的¹。虽然导致胎盘生理异常的靶向小鼠突变的数量继续增加², 但目前在小鼠基因组信息学 (凡陀) 网站上列出的219种基因型是 (http://www.informatics.jax.org/vocab/mp_ontology/MP:0010038), 许多这些表型从机械的角度看不清楚。除了基因改变, 小分子药物, 单克隆抗体, 毒素, 病原体, 过量代谢物, 或各种环境因子可能影响胎盘发育和功能^3,4,5,6,7. 然而, 简单的体外分析了胎盘发育中的模型关键步骤是稀缺的。

小鼠胎盘发育在早期妊娠开始, 当尿 (脐带的发育前体) 从胚的后端出现, 作为一个芽, 生长向绒毛膜的⁸。Chorioadhesive 细胞在囊芽的外层斡旋在绒毛膜 (囊 “融合”) 的表面上的附着和传播尿。绒毛膜随后折叠成绒毛进入其中尿的血管生长形成迷宫, 内部胎盘层在那里营养和氧气被交换在严密并列的母体血液窦和胚胎血管之间^{9 ,10}。

尿对绒毛膜的附着是迷宫形成的初始和关键步骤, 而这一过程中的缺陷是妊娠¹中最常见的胚胎致死原因之一。虽然已经描述了许多鼠标突变体, 但囊附件无法出现在¹⁰, 并且凡陀数据库当前列出了108个以异常囊融合为特征的鼠标突变体 (http://www.informatics.jax.org/vocab/mp_ontology/MP:0002824), 血管细胞黏附分子之间的相互作用 molecule-1 (VCAM-1, 表达囊胚层) 和α4β1整合素 (表达在绒毛膜皮, 这是派生自外胚层) 似乎是必不可少的囊附件和融合^11,12,13。全芒野型胚的免疫组织化学染色表明, VCAM-1 在囊茎远端2/3 处以大约胚胎日 (E) 7.5¹³ (1-4 节阶段) 表达, 其表达仍然高在囊附件和-融合^11,12期间。囊附着于绒毛膜的失败通常通过子宫芽的组织学分析来检测。然而, 尿大小是生理上高度可变的在之间, 甚至在同一发育阶段的凋落物¹⁴, 和尿只能附加到绒毛膜当它已经达到足够的大小, 使身体接触。反射这自然可变性, 囊附件发生在 ~ e 8.0 和 e 9.0在子宫内, 并且一个统计地可靠评估这个过程由组织学是依赖于分析许多conceptuses 在适当的发育阶段获得足够的标本。

在这里, 我们描述一种方法来评估囊附件前子宫, 不太依赖于尿大小。我们展示了小鼠胚胎的解剖和他们的 allantoides 从怀孕的老鼠的子宫〜8天后 coitum (dpc; dpc 0.5 指定阴道插头的检测), 并随后培养尿外植体的固定α4β1整合.该方法能够对多尿外植体在不同的连续时间点上的附着和扩展行为进行快速、实用的评价。该协议可用于筛选目标突变、药物或各种环境因素对尿附着体的影响ex 体内。

Protocol

小鼠育种得到了 Regierung Unterfranken 的批准, 所有的分析都严格按照德国和欧洲联盟关于实验室动物护理和使用的法律和法规进行。 1. 尿外植体培养的α4β1整合素孔板包衣 重组冻干小鼠α4β1整合素在200µg/毫升无菌磷酸盐缓冲盐水 (PBS)。在 5ml (37 ° c) PBS 中稀释至最终浓度为10µg/毫升。吸管20µL/稀释α4β1整合素溶液到所需数量的孔板 (1 尿/井) 的井。注: 在孔板上?…

Representative Results

本协议描述了一种方法来隔离和外植体小鼠 allantoides前体, 并详细的定性评估的尿附着α4β1整合素, 一个关键的过程中, 在体内囊融合。在图 1A-H中显示的代表性结果演示了从怀孕子宫开始的尿隔离的连续步骤。围绕子宫角的母体组织, 如脂肪组织和血管 (图 1A) 被移除, 并在打开肌肉子宫层?…

Discussion

在血清的存在, 这是一个丰富的来源, pro-adhesive 细胞外基质分子, 如纤维连接蛋白, 尿植体将欣然附加到各种塑料, 玻璃, 或过滤表面^9,16。因此, 如果进行的试验的目的是专门研究基因修饰或任何其他治疗对固定化α4β1尿附着的影响, 就必须阻止非特异性结合位点 (例如, 用牛血清白蛋白) 涂层后的表面与整合素和前孵化与含血清的媒体。

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Materials

C57BL/6J wildtype mice	The Jackson Laboratory	Stock No: 000664
70 % (v/v) Ethanol	VWR International	930031006
Standard pattern forceps	Fine Science Tools	11000-12	Forceps used to open the abdominal cavity.
Micro dissecting forceps	Fine Science Tools	11254-20	Dumont # 5 medical biology forcepts with fine, sharp tip.
Fine scissors	Fine Science Tools	14094-11	Straight blades.
Spring scissors	Fine Science Tools	15012-12	Noyes spring scissors with 14 mm blades for the dissection of uterus buds.
Microspoon	Carl Roth	AT18.1	5 mm Spoon diameter.
Microtiter plates	ibidi	81501	We use uncoated, sterile angiogenesis slides (internal volume 50 µL) with a hydrophobic surface that is not tissue-culture treated to reduce non-a4b1-mediated binding to plastic.
10 cm dish	Nunc	150350	Sterile tissue culture dishes.
3.5 cm dish	Nunc	150288	Sterile tissue culture dishes.
Large orifice pipette tips	Biozym	VT0140X	Low binding pipette tips, 200 µL.
a4b1 integrin	R&D Systems	6054-A4	Murine a4b1 integrin recombinantly expressed and purified from a Chinese hamster ovary cell line.
Dulbecco's Phosphate Buffered Saline (PBS)	Sigma Aldrich	D8537	Without Ca2+ and Mg2+.
Dulbecco’s Modified Eagle Medium (DMEM)	PAN Biotech	P04-03600	The formulation contains 4.5 g/L glucose, 110 mg/L sodium pyruvate and 3.7 g/L NaHCO3 but no L-glutamine, which is added separately.
Penicillin/Streptomycin	Gibco (ThermoFisher Scientific)	15140-122	100 x (10,000 U/mL Penicillin and 10,000 µg/mL streptomycin) stock solution. Prepare and store aliquots at -20 °C to avoid freeze/thaw.
L-Glutamine	PAN Biotech	P04-80100	100 x (200 mM) Stock solution. Prepare and store aliquots at -20 °C to avoid freeze/thaw.
Fetal bovine serum	Biochrom	S 0115	We use heat-inactivated FBS (heated to 56 °C for 30 min with mixing to inactivate complement).
Bovine serum albumin (BSA)	Sigma Aldrich	A7030	We use protease- and fatty acid-free albumin. Prepare a 0.1 % (w/v) solution in DMEM. Sterile filter the solution through a disposable cell culture filter with a 0.22 µm pore size and low protein-binding membrane, and store aliquots at -20 °C.
para-Formaldehyde	Carl Roth	0335.2	To make a formaldehyde solution in PBS, weigh para-formaldehyde powder in a ventilated hood, and add it to PBS preheated to ca. 60 °C in a beaker on a stir plate. Add 1 N NaOH dropwise until solution clears. Let solution cool to room temperature, and filter through 0.22 mm filter syringe. Adjust pH with diluted HCl to ca. 6.9, and adjust to final volume with PBS. We aliquot and freeze the solution, but it can also be stored at 4 °C for approximately four weeks.
Triton X-100	Sigma Aldrich	X100	Use wide-orifice pipette tips to handle undiluted Triton X-100.
Normal goat serum	Sigma Aldrich	G9023	To block non-specific binding of antibodies in immunofluorescence analyses.
a-CD31 Antibodies	BD Biosciences	550274	Purified rat anti-mouse monoclonal antibodies, clone MEC 13.3.
Secondary goat anti-rat antibodies	Thermo Fisher	A-11006	Goat anti-rat IgG (heavy and light chains), cross-adsorbed, fluorescently labeled with Alexa Fluor 488. Other fluorescent labels are also possible.
Mowiol 4-88	Sigma Aldrich	81381	Mounting medium for cytochemistry. MW ~31,000
Labovert	Leitz	Labovert	Inverted phase contrast microscope equipped with a 4 x and 10 x objective for somite pair counting. Other phase contrast microscopes (such as those that are routinely used in tissue culture) are also suitable.
M80	Leica Microsystems	M80	Stereomicroscope with 8 : 1 zoom range (yielding a magnification between 7.5 x and 60 x) for embryo and allantois dissection.
DM4000B	Leica Microsystems	DM4000B	Microscope system for histology with LED illumination. We use HCX PL FLUOTAR 5 x/0.15 and HC PL FLUOTAR 10 x/0.30 objectives. Other microscopes equipped phase contrast and fluorescence optics can be used to score allantois attachment.
Digital camera	JVC	KY-F75U	3-CCD digital capture camera attached to the DM4000B LED microscope. We use Diskus software (Hilgers) to operate both the microscope and the camera.
Confocal microscope	Leica Microsystems	SP5	Confocal microscope for higher-resolution imaging of endothelia in the vascular plexus of allantois explants. Immunofluorescence images were taken with a 40 x objective.