主动脉-性腺-肾外植体培养体系在发育性造血中的应用
Summary
本议定书描述使用培养的主动脉-性腺-肾的表达分析, 菌落形成单位在文化和脾脏, 并长期重建, 以确定影响的调节因子和信号通路的造血干细胞发育。这已被证明是一个有效的系统研究造血干细胞生物学和功能。
Abstract
使用小鼠胚胎进行造血研究的局限性是在手术中增加了不便, 这主要是由于胚胎的宫内发育。尽管来自击倒 (ko) 小鼠的基因数据令人信服, 但在需要时为所有基因生成高鼠并不现实。此外, 执行在体内抢救实验, 以巩固从 KO 小鼠获得的数据是不方便的。为了克服这些局限性, 主动脉-性腺-肾 (年会) 外植体培养作为研究造血干细胞 (HSC) 发育的适宜系统而发展起来。特别是在抢救实验中, 可用于恢复高鼠的造血损伤。通过在培养基中加入适当的化学物质, 可以重新激活受损的信号, 或抑制上调的通路。利用这种方法, 可以进行大量的实验, 以确定 hsc 发展的关键调控因子, 包括 hsc 相关基因在 mRNA 和蛋白质水平、菌落形成能力和重构能力上的表达。这一系列的实验将有助于确定哺乳动物 HSC 发育所必需的基本机制。
Introduction
造血干细胞 (干细胞) 是组织特异的成体干细胞, 具有多向的潜能, 包括红、髓细胞和淋巴细胞, 以及自我的能力。最近的研究表明, 最早的干细胞产生于一个专门的内皮细胞, 称为 hemogenic 内皮细胞 (他), 通过内皮到造血转移 (EHT) 在腹壁的背主动脉1,2 ,3,4。一旦从胚胎 (E) 10.5 到 E12.5 在小鼠胚胎中形成了主动脉-性腺-肾 (年会) 区域, 干细胞就会迁移到胎儿肝脏进行扩张, 最终在个体的生命中殖民骨髓以维持成人造血。5,6. 尽管这一研究已经进行了许多年, 但 HSC 产生和发展的基本机制仍未得到完全理解。
与体外不同的是, 斑马鱼胚胎的受精和发育, 小鼠胚胎的宫内发育使得在胚胎发生过程中的最终造血研究更不方便。虽然使用击倒 (ko) 小鼠的基因实验是常用的, 但某些高鼠的缺乏也限制了它们在造血研究领域的应用。此外,在体内抢救实验在 KO 小鼠中不易进行。自1996年以来, 在该领域的先驱在7中为造血研究开发了 “年度” 外植体培养。在这一文化体系的帮助下, 已经确定了股东大会区域的腹侧组织以促进 HSC 活动, 而背部组织则发挥相反的作用8,9。在 HSC 的发展中, 应用了 Mpl、蛋白、蛋白、BMP 和刺猬的信号, 确定了血清素、外植体培养系统的作用10、11、12、13、14. 重要的是, 它也是一种常用的方法来挽救突变体胚胎的造血缺损13,15。
Protocol
Representative Results
Discussion
众所周知, 成熟的干细胞在骨髓中可以重新填充受照射的接受者的血液系统。与这些功能干细胞不同的是, 在小鼠胚胎的周年大会上出现的干细胞是不成熟的。直接移植结果表明, I 型 (ve cad+ CD45– CD41+) 和类型 II (ve cad+ CD45+) 前干细胞具有没有重构能力20。利用股东大会的外植体培养体系, 在干细胞的新生中, 可以在移植试验中重建受体<sup clas…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢普华永在图准备方面的帮助。这项工作得到了中国国家自然科学基金 (81530004、31425016) 和中国科学技术部 (2016YFA0100500) 的资助。. 进行了实验并草拟了手稿;–编辑了手稿。两位作者阅读并批准了最后的手稿。
Materials
| durapore 0.65 µm filters | Millipore | R5BA63787 | AGM explant culture |
| M5300 long-term culture medium | Stem Cell Technologies | 5300 | AGM explant culture |
| Trizol | Tiangen | 5829 | RNA extration |
| M-MLV reverse transcriptase | Promega | 90694 | reverse transcription |
| SYBR Green | Qiagen | A6002 | quantatitive real-time PCR |
| protease inhibitor | Roche | 55622 | Protein extration |
| collagenase | Sigma | C2674 | digestion of AGM tissues |
| MethoCult GF M3434 medium | Stem Cell Technologies | 3434 | CFU-C assay |
| ultra-low attachment 24-well plates | Costar | 3473 | CFU-C assay |
| C57BL/6 CD45.2 mice | Beijing HFK Bioscience Co. Ltd | CFU-S assay | |
| C57BL/6 CD45.1 mice | Beijing HFK Bioscience Co. Ltd | Long-term transplantation | |
| phosphate buffered saline | Life Technologines | 8115284 | AGM Collection |
| Penicillin-Streptomycin solution | HyClone | SV30010 | AGM explant culture |
| anti-CD45.2-PE-CY7 | eBioscience | 25-0454-80 | Long-term transplantation |
| anti-CD45-FITC | eBioscience | 11-0451-81 | Long-term transplantation |
| UV lamp | Beijing jiangmorning yuan bio-technology co., LTD | 039-14402 | power: 30W |
| stainless steel mesh | AS ONE SHANGHAI Corporation | 2-9817-10 | aperture diameter: 2.5 mm |
| hydrocortisone | Sigma | H0396 | selectively diluted into M5300 medium |
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