小鼠胰腺内分泌细胞的单细胞 Transcriptomic 分析
Summary
我们描述了一种方法, 从胚胎, 新生儿和产后胰腺的内分泌细胞分离, 其次是单细胞 RNA 测序。这种方法可以分析胰腺内分泌谱系的发展, 细胞异质性和 transcriptomic 动力学。
Abstract
胰腺内分泌细胞聚集在胰岛, 调节血糖稳定性和能量代谢。胰岛细胞的不同类型, 包括胰岛素分泌β细胞, 在胚胎阶段与常见的内分泌祖先分化。未成熟的内分泌细胞通过细胞增殖和成熟在长时间的产后发育期内扩张。然而, 这些进程所依据的机制并没有明确界定。单细胞 RNA 测序是一种很有希望的方法来表征不同的细胞种群和追踪细胞谱系分化途径。在这里, 我们描述了从胚胎, 新生儿和产后胰腺分离的胰β细胞单细胞 RNA 序列的方法。
Introduction
胰腺是哺乳动物重要的代谢器官。胰腺由内分泌和外分泌的隔间组成。胰腺内分泌细胞, 包括产生胰岛素的β细胞和胰高血糖素生成α细胞, 聚集在胰岛并协调调节系统葡萄糖稳态。内分泌细胞功能失调导致糖尿病, 这已成为世界上一个重要的公共卫生问题。
胰腺内分泌细胞在胚胎发生1期间由 Ngn3+祖祖先衍生。后期, 在围产期, 内分泌细胞增殖形成未成熟的胰岛。这些不成熟的细胞继续发展, 逐渐成为成熟的胰岛, 这成为丰富的血管化调节血糖动态平衡在成人2。
虽然已经确定了一组转录因子来调节β细胞分化, 但β细胞的精确成熟途径还不清楚。此外, β细胞的成熟过程也涉及到细胞数量扩展3,4和细胞异质性的生成5,6。然而, 这些过程的监管机制还没有得到很好的研究。
单细胞 RNA 测序是一种强有力的方法, 可以分析细胞亚群和跟踪细胞谱系发展途径7。利用这一技术, 在胰岛发育过程中发生的关键事件可以在单细胞8级破译出来。在单细胞 RNA 测序协议中, Smart-seq2 允许生成全长 cDNA, 提高灵敏度和准确度, 并使用标准试剂降低成本9。Smart-seq2 大约需要两天时间来构建一个用于序列10的 cDNA 文库。
在这里, 我们提出了一种方法, 从胎儿胰腺的荧光标记β细胞分离到成年 Ins1-RFP 转基因小鼠11, 使用荧光活化细胞分类 (资产管制署), 并在 transcriptomic 分析的性能单细胞水平, 使用 Smart-seq2 技术 (图 1)。该协议可推广到所有胰腺内分泌细胞类型在正常、病理和衰老状态下的 transcriptomes 分析。
Protocol
这里所描述的所有方法都已获得北京大学机构动物护理和使用委员会 (IACUC) 的批准。 1. 胰腺隔离 为 E17.5 (胚胎天 17.5) 胚胎: 估计胚胎日0.5 根据时间点时, 阴道插头出现。 通过2的管理, 牺牲怀孕的老鼠。用70% 酒精喷雾腹部毛皮。 用剪刀从延伸到肋骨的生殖器区做一个 V 形切口。这个过程将完全打开腹腔。 解剖子宫…
Representative Results
胰腺从胚胎, 新生儿和产后小鼠 (图 2A 和2B) 进行解剖。对于18岁以上的小鼠, 消化作用取决于灌注程度;因此, 注射是胰岛隔离的最重要步骤 (图 2C-2E和表 6)。在这一步中, 尽可能多地注射胶原酶来填充胰腺。完全膨胀的胰腺如图 2D所示。如果灌注不成功 (<st…
Discussion
在本协议中, 我们展示了一种有效且易于使用的方法来研究胰β细胞的单细胞表达谱。该方法可用于分离胚胎、新生儿和产后胰腺的内分泌细胞, 并进行单细胞 transcriptomic 分析。
最关键的步骤是在良好的条件下隔离单个β细胞。充分灌注胰腺对后续消化反应较好。灌注不足, 通常发生在背胰, 将导致低胰岛产量。灌注后, 消化时间和晃动强度需要特别注意。过度消化, 由于长时间…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢国家蛋白质科学中心, 北京 (北京大学) 和北京-清华生命科学计算平台中心。这项工作得到中国科学技术部 (2015CB942800)、中国国家自然科学基金 (31521004、31471358和 31522036) 的支持, 并由北京-清华大学生命科学中心向 C. R.X. 提供资助。
Materials
| Collagenase P | Roche | 11213873001 | |
| Trypsin-EDTA (0.25 %), phenol red | Thermo Fisher Scientific | 25200114 | |
| Fetal bovine serum (FBS) | Hyclone | SH30071.03 | |
| Dumont #4 Forceps | Roboz | RS-4904 | |
| Dumont #5 Forceps | Roboz | RS-5058 | |
| 30 G BD Needle 1/2" Length | BD | 305106 | |
| Stereo Microscope | Zeiss | Stemi DV4 | |
| Stereo Fluorescence microscope | Zeiss | Stereo Lumar V12 | |
| Centrifuge | Eppendorf | 5810R | |
| Centrifuge | Eppendorf | 5424R | |
| Polystyrene Round-Bottom Tube with Cell-Strainer Cap | BD-Falcon | 352235 | |
| 96-Well PCR Microplate | Axygen | PCR-96-C | |
| Silicone Sealing Mat | Axygen | AM-96-PCR-RD | |
| Thin Well PCR Tube | Extragene | P-02X8-CF | |
| Cell sorter | BD Biosciences | BD FACSAria | |
| Capillary pipette | Sutter | B100-58-10 | |
| RNaseZap | Ambion | AM9780 | |
| ERCC RNA Spike-In Mix | Life Technologies | 4456740 | |
| Distilled water | Gibco | 10977 | |
| Triton X-100 | Sigma-Aldrich | T9284 | |
| dNTP mix | New England Biolabs | N0447 | |
| Recombinant RNase Inhibitor | Takara | 2313 | |
| Superscript II reverse transcriptase | Invitrogen | 18064-014 | |
| First-strand buffer (5x) | Invitrogen | 18064-014 | |
| DTT | Invitrogen | 18064-014 | |
| Betaine | Sigma-Aldrich | 107-43-7 | |
| MgCl2 | Sigma-Aldrich | 7786-30-3 | |
| Nuclease-free water | Invitrogen | AM9932 | |
| KAPA HiFi HotStart ReadyMix (2x) | KAPA Biosystems | KK2601 | |
| VAHTS DNA Clean Beads XP beads | Vazyme | N411-03 | |
| Qubit dsDNA HS Assay Kit | Invitrogen | Q32854 | |
| AceQ qPCR SYBR Green Master Mix | Vazyme | Q121-02 | |
| TruePrep DNA Library Prep Kit V2 for Illumina | Vazyme | TD502 | Include 5x TTBL, 5x TTE, 5x TS, 5x TAB, TAE |
| TruePrep Index Kit V3 for Illumina | Vazyme | TD203 | Include 16 N6XX and 24 N8XX |
| High Sensitivity NGS Fragment Analysis Kit | Advanced Analytical Technologies | DNF-474 | |
| 1x HBSS without Ca2+ and Mg2+ | 138 mM NaCl; 5.34 mM KCl 4.17 mM NaHCO3; 0.34 mM Na2HPO4 0.44 mM KH2PO4 |
||
| Isolation buffer | 1 × HBSS containing 10 mM HEPES, 1 mM MgCl2, 5 mM Glucose, pH 7.4 | ||
| FACS buffer | 1 × HBSS containing 15 mM HEPES, 5.6 mM Glucose, 1% FBS, pH 7.4 | ||
| NaCl | Sigma-Aldrich | S5886 | |
| KCl | Sigma-Aldrich | P9541 | |
| NaHCO3 | Sigma-Aldrich | S6297 | |
| Na2HPO4 | Sigma-Aldrich | S5136 | |
| KH2PO4 | Sigma-Aldrich | P5655 | |
| D-(+)-Glucose | Sigma-Aldrich | G5767 | |
| HEPES | Sigma-Aldrich | H4034 | |
| MgCl2 | Sigma-Aldrich | M2393 | |
| Oligo-dT30VN primer | 5'-AAGCAGTGGTATCAACGCAGAGTACT30VN-3' | ||
| TSO | 5'-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3' | ||
| ISPCR primers | 5'-AAGCAGTGGTATCAACGCAGAGT-3' | ||
| Gapdh Forward primer | 5'-ATGGTGAAGGTCGGTGTGAAC-3' | ||
| Gapdh Reverse primer | 5'-GCCTTGACTGTGCCGTTGAAT-3' | ||
| Ins2 Forward primer | 5'-TGGCTTCTTCTACACACCCA-3' | ||
| Ins2 Reverse primer | 5'-TCTAGTTGCAGTAGTTCTCCA-3' |
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Citar este artigo
Li, L., Yu, X., Zhang, Y., Feng, Y., Qiu, W., Xu, C. Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells. J. Vis. Exp. (139), e58000, doi:10.3791/58000 (2018).