使用体内和组织和细胞外植体方法研究胚胎和围产期主动脉的形态和发病机制
Summary
用于研究胚胎和围产期小鼠主动脉的协议使用在体内克隆分析和命运图, 主动脉外植体和孤立平滑肌细胞在这里详述。这些不同的方法有助于调查胚胎和围产期主动脉在正常发育和疾病的发病机制。
Abstract
主动脉是体内最大的动脉。主动脉壁由内皮细胞内层、交变弹性板中层和平滑肌细胞 (校董)、外层成纤维细胞和胞外基质组成。与广泛研究的病理模型 (例如,动脉粥样硬化) 在成人主动脉, 更少的是已知的胚胎和围产期主动脉。在这里, 我们专注于校董, 并提供协议, 分析胚胎和围产期主动脉校董在正常发育和疾病的发生和发病机制。具体来说, 包括的四项协议是: i)在体内胚胎的命运映射和克隆分析;二) 外植体胚胎主动脉培养;三) SMC 与围产期主动脉隔绝;和 iv) 皮下渗透 mini-pump 放置在怀孕 (或非) 小鼠。因此, 这些方法有助于调查的起源 (s), 命运, 和克隆结构的校董在主动脉在体内。他们允许通过持续接触药理学药物来调节胚胎主动脉形态的在子宫内。此外, 孤立的主动脉组织外植体或主动脉校董可用于在诸如 muscularization、增殖和迁移等基本过程中获得对特定基因靶点作用的洞察。这些假说生成实验的孤立校董和说明主动脉, 然后可以评估在体内的情况下, 通过药理和遗传方法。
Introduction
多细胞生物体的循环系统功能向不与外界环境接触的细胞输送养分和氧气, 并从这些细胞中去除废物和二氧化碳。在脊椎动物中, 初级循环系统由心脏组成, 它通过一系列血管泵血。大血管的壁, 如动脉和静脉, 由三层组成: i) 内皮细胞内膜, 或内部层;2) 培养基, 或中间层交替圆周细长平滑肌细胞校董和弹性片状;和 iii) 的膜, 或外层结缔组织和成纤维细胞。绝大多数的血管生物学研究集中在内皮细胞, 通过血管生成研究新的内皮细胞内衬管的形成。相比之下, 校董得到的关注相对较少。然而, 校董是一个关键的细胞类型, 在建设正常动脉壁和血管病理。
主动脉是体内最大口径的动脉, 接受心脏左心室的输出。它受到各种人类疾病的折磨, 包括动脉粥样硬化、动脉瘤和解剖。在成人机体中, 主动脉及其主要分支在血管疾病模型中得到了深入的研究。例如, 高脂肪饮食喂养小鼠是无效的基因编码低密度脂蛋白受体或载脂蛋白 E, 发展动脉粥样硬化, 和最近的命运测绘研究表明, 预先存在的校董产生多种细胞类型的动脉粥样硬化斑块1。在主动脉瘤中, 病理改变包括 SMC 细胞凋亡和细胞外基质重塑2,3。
在胚胎和围产期期间, 对 SMC 的形态发生和发病机制的认识显著减少。在这里, 我们提供了研究胚胎和围产期主动脉校董在体内, 组织外植体和孤立细胞的协议。例如, 该协议的第一部分揭示了胚胎小鼠的命运映射和克隆分析。重组在细胞特异启动子的控制下表达, 促进特定细胞及其子代的标记4,5,6;然而, 在小鼠胚胎发育过程中, 对细胞特异标记的时间控制可能具有挑战性。在这方面, 在校董 (例如, Myh11或Acta2) 和一个综合招聘报告员的 CreER 下, 我们提供了在怀孕的水坝中注射他莫昔芬或其活性代谢物 4-OH-苯氧胺的方法。用于分析胚胎或产后后代的标记细胞。此外, 与命运测绘研究, 这是以一个单一的记者荧光1,7, 克隆分析大大加强与多色综合招聘记者。
第二和第三部分的协议描述的方法, 分离和培养胚胎主动脉外植体和主动脉校董的新生儿, 分别。这些方法允许操纵信号通路, 特别是在主动脉外植体或校董, 并分析药物的直接作用。因此, 在兴趣组织中的特定基因的作用可以比传统的小鼠遗传操作更快速地进行筛选。此外, 孤立的 SMC 研究有助于分析细胞迁移和黏附, 这是技术上有限的在体内。
最后, 第四议定书部分划定了在怀孕 (或非) 小鼠体内装有药理剂的皮下渗透 mini-pump 的位置。这种方法有助于分析由于新陈代谢快而需要连续输液的药剂对胚胎发育的影响。经常注射的替代品对许多药物来说是不可行的, 应该避免, 因为它可能会在怀孕的大坝中造成严重的不适。
Protocol
Representative Results
Discussion
与大鼠主动脉及其主要分支在成人病理条件下, 如动脉粥样硬化模型的广泛研究不同, 对胚胎和围生期主动脉的形态发生和发病机制的了解较少。在这里, 我们专注于胚胎/围产期主动脉, 特别是校董, 并提供协议, 以研究主动脉通过在体内, 组织外植体, 和 SMC 隔离方法。这些免费的方法为研究者提供了不同的方法来研究胚胎/围产期主动脉。
克隆分析是一种非常强大的方法…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢李院长分享他的实验室的主动脉 SMC 隔离协议。资助由国立卫生研究院 (R21NS088854、R01HL125815 和 R01HL133016 博士 G)、美国心脏协会 (补助金14GRNT19990019 至 D.M.G.) 和耶鲁大学 (布朗-柯克斯奖学金提供给上午和启动资金 D.M.G.)。
Materials
| Tamoxifen | Sigma | T5648 | |
| Corn oil | Sigma | C-8267 | Vehicle for tamoxifen |
| 4-OH-tamoxifen | Sigma | H7904 | Active metabolite of tamoxifen |
| Progesterone | Sigma | P8783-5G | Use at half the concentration of tamoxifen |
| OCT compound | Sakura tissue tek | 4583 | For making cryoblocks |
| Cryomolds | Polysciences inc | 18986 | |
| DAPI | Sigma | D9542 | IHC staining of nucleus, final concentration 5 mg/ml |
| Cy3 directly conjugated anti-SMA antibody | Sigma | A2547 | IHC staining of SMA, final dilution 1:500 |
| Anti-CD31 antibody | BD Pharmingen | 550274 | IHC staining of GFP, final concentration 0.006 mg/ml |
| Anti-GFP antibody | Thermo Fisher Scientific | A-11121 | IHC staining of CD31, final concentration 0.0016 mg/ml |
| Secondary antibody goat anti-rabbit, Alexa 647 | Life Technologies | a21244 | IHC staining, final concentration 0.004 mg/ml |
| Secondary antibody goat anti-rabbit, Alexa 488 | Life Technologies | a11008 | IHC staining, final concentration 0.004 mg/ml |
| DMEM | Thermo Fisher Scientific | 10567-014 | For cell culture |
| FBS | Thermo Fisher Scientific | 10437028 | |
| Anti-integrin beta3 blocking antibody | BD Biosciences | 553343 | Clone 2C9.G2, final concentration 0.02 mg/ml |
| Collagenase | Worthington Biochemical Corp | 44H14977A | For digesting aorta |
| Elastase | Worthington Biochemical Corp | 34K15139 | For digesting aorta |
| Antibiotic-antimycotic (100X) | Thermo Fisher Scientific | 15240062 | |
| Recombinant human FGF | Promega | G5071 | |
| Recombinant human EGF | Promega | G5021 | |
| Penicillin/streptomycin (10,000 U/ml) | Thermo Fisher Scientific | 15140122 | |
| Amphotericin B | Thermo Fisher Scientific | 15290026 | |
| Tissue culture plates | Corning | CLS430165 | |
| Alzet osmotic mini-pump | Durect Corporation | 2001 | |
| ECLIPSE 80i Upright Fluorescent Microscope | Nikon | ||
| TCS SP5 | Leica | ||
| Branson Sonifier 450 | VWR | ||
| Myh11-CreERT2 mice | The Jackson Laboratory | 19079 | |
| Acta2-CreERT2 mice | Obtained from lab of Dr. Pierre Chambon and Daniel Metzger | ||
| ROSA26R-CreERT2 mice | The Jackson Laboratory | 8463 | |
| ROSA26R(mTmG/mTmG) mice | The Jackson Laboratory | 026862 | |
| ROSA26R(EYFP/EYFP) mice | The Jackson Laboratory | 006148 | |
| ROSA26R(Confetti/Confetti) mice | The Jackson Laboratory | 13731 | |
| ROSA26R(Rb/Rb) mice | Lab of Dr. Irv Weissman | Obtained from lab of Dr. Irv Weissman |
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