通过细胞外囊泡/外来体转移乳腺基质上皮细胞和乳腺腔细胞之间的乳腺形成能力
Summary
该方案描述了从非贴壁/间充质乳腺上皮细胞中纯化，定量和表征细胞外囊泡（EVs）/外来体的方法，并使用它们将乳腺形成能力转移到乳腺上皮细胞。衍生自茎状乳腺上皮细胞的EVs /外来体可以将该细胞特性转移到摄入EV /外来体的细胞中。
Abstract
细胞可以通过外来体，含有蛋白质，脂质和核酸的〜100-nm胞外囊泡（EVs）进行通信。非贴壁/间充质乳腺上皮细胞（NAMEC）衍生的细胞外囊泡可以通过差异超速离心从NAMEC培养基中分离。基于它们的密度，EV可以通过110,000×g的超速离心纯化。可以使用连续密度梯度进一步分离来自超速离心的EV制剂，以防止可溶性蛋白质的污染。然后可以使用纳米颗粒跟踪分析进一步评估纯化的EV，其测量制剂中囊泡的大小和数量。尺寸范围为50至150nm的细胞外囊泡是外来体。 NAMEC衍生的EVs /外来体可以通过乳腺上皮细胞摄取，可以通过流式细胞术和共焦显微镜检测。一些乳腺干细胞性质（ 如乳腺形成能力）可以通过来自NAMEC的EVs /外来体从茎状NAMEC转移到乳腺上皮细胞。分离的原代EpCAM hi / CD49f细胞乳腺上皮细胞在移植到小鼠脂肪垫后不能形成乳腺，而EpCAM lo / CD49f基底乳腺上皮细胞在移植后形成乳腺。通过EpCAM hi / CD49f细胞乳腺上皮细胞摄取NAMEC衍生的EVs /外来体允许它们在移植到脂肪垫中之后产生乳腺。衍生自干样乳腺上皮细胞的EVs /外来体转移到EpCAM hi / CD49f细胞乳腺上皮细胞的乳腺形成能力。
Introduction
外来体可以通过在细胞之间转移膜和细胞质蛋白，脂质和RNA来介导细胞交流1 。已经证明外来体介导的通信涉及许多生理和病理过程（ 即抗原呈递，耐受性发展2和肿瘤进展3 ）。外来体通常具有与释放它们的源细胞相似的内容。因此，外来体可以从源细胞携带特定的细胞特性，并将这些性质转移到摄取它们的细胞中。
外来体是50至150纳米的双层膜囊泡，并呈现特异性标记（ 例如 CD9，CD81，CD63，HSP70，Alix和TSG101）。因此，外来体必须通过不同方面的各种方法来表征。透射电子显微镜可用于观察膜囊泡如外来体4,5 。纳米粒子跟踪分析（NTA）和动态光散射分析（DLS）用于测量纯化外来体的大小和数量4 。可以通过密度梯度验证外来体的脂质膜含量。染色体标志物，例如CD9，CD81，CD63，HSP70，Alix和TSG101,6,7可以通过Western印迹法测定。
乳腺基底细胞在植入脂肪垫时具有产生乳腺的能力，而腔细胞不能8,9,10 。因此，乳腺基底细胞也称为乳腺再生单位。通过使用乳腺基底细胞和腔细胞模型，可以检测EVs /外来体在不同细胞群体之间转运细胞特征的能力。这个工作证明了通过使用源自乳腺基底上皮细胞的EVs /外来体将腺体形成能力从乳腺基底上皮细胞转移到乳腺腔上皮细胞的方法。乳腺上皮细胞摄取基底细胞分泌的EV /外来体后获得基础细胞特性，然后形成乳腺。
Protocol
所有涉及动物的研究均符合动物保护机构委员会批准的方案。 细胞外囊泡/外来体分离和鉴定培养乳腺上皮基底细胞NAMEC4，用500mL MCDB 170，pH 7.4 + 500mL DMEM / F12与碳酸氢钠（0.2438％）制备的新鲜无血清培养基; EGF（5 ng / mL）;氢可替康（0.5μg/ mL）;胰岛素（5μg/ mL）;牛垂体提取物（BPE;35μg/ mL）;和GW627368X（1μg/ mL）。 用血细胞计数器计数细胞后，在第0天将1.2×10 6?…
Representative Results
由于已经显示阻断PGE 2 / EP 4信号传导从乳腺基底样干细胞4引发EV /外来体释放，这项工作提出了从乳腺上皮基底细胞（NAMEC）培养物中分离诱导的EVs /外来体的方法。由于NAMECs在无血清培养基中培养，所以不存在来自血清13的预先存在的EV /外来体。对于在含有血清的培养基中培养的细胞，培养基中预先存在的外来体必须通…
Discussion
外来体通常携带释放它们的细胞的特征，并且释放的外来体的量可以由刺激诱导4 。可以收集细胞的培养基并进行差异超速离心用于EV /外来物质收集（ 图1 ）。目前对于分离EV /外来体的理想方法目前尚无普遍的共识。这里使用的最佳方法已经由下游应用14确定 。超速离心是分离EV /外来体的相对快速的方法，可以保护EV /外来体的生物活性?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生研究机构（05A1-CSPP16-014，HJL）和科技部（MOST 103-2320-B-400-015-MY3，HJL）的资助。
Materials
| MCDB 170 | USBiological | M2162 | |
| DMEM/F12 | Thermo | 1250062 | |
| Optima L-100K ultracentrifuge | Beckman | 393253 | |
| SW28 Ti Rotor | Beckman | 342204 | |
| SW41 Rotor | Beckman | 331306 | |
| NANOSIGHT LM10 | Malvern | NANOSIGHT LM10 | for nanoparticle tracking analysis (NTA) |
| Optiprep | Sigma-Aldrich | D1556 | 60% (w/v) solution of iodixanol in water (sterile). |
| CD81 antibody | GeneTex | GTX101766 | 1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4°C, overnight |
| CD9 antibody | GeneTex | GTX100912 | 1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4°C, overnight |
| CD63 antibody | Abcam | Ab59479 | 1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4°C, overnight |
| TSG101 antibody | GeneTex | GTX118736 | 1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4°C, overnight |
| GAPDH | GeneTex | GTX100118 | 1:6000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4°C, overnight |
| CFSE (carboxyfluorescein succinimidyl diacetate ester) | Thermo | V12883 | |
| FACSCalibur | BD Biosciences | fluorescence cell analyzer | |
| collagenase Type IV | Thermo | 17104019 | |
| trypsin | Thermo | 27250018 | |
| ITS | Sigma-Aldrich | I3146 | a mixture of recombinant human insulin, human transferrin, and sodium selenite |
| accutase | ebioscience | 00-4555-56 | a natural enzyme mixture with proteolytic and collagenolytic enzyme activity |
| dispase | STEMCELL | 7913 | 5 mg/ml = 5 U/ml |
| anti-CD49f antibody | Biolegend | 313611 | 1:50 |
| anti-EpCAM antibody | Biolegend | 118213 | 1:200 |
| FACSAria | BD Biosciences | cell sorter | |
| carmine alum | Sigma-Aldrich | C1022 | |
| human mammary epithelial cells (HMLE cells, NAMECs) | gifts from Dr. Robert Weinberg | ||
| permount | Thermo Fisher Scientific | SP15-500 | |
| sodium bicarbonate | Zymeset | BSB101 | |
| EGF | Peprotech | AF-100-015 | |
| Hydrocoritisone | Sigma-Aldrich | SI-H0888 | |
| Insulin | Sigma-Aldrich | SI-I9278 | |
| BPE (bovine pituitary extract) | Hammod Cell Tech | 1078-NZ | |
| GW627368X | Cayman | 10009162 | |
| 15-cm culture dish | Falcon | 353025 | |
| table-top centrifuge | Eppendrof | Centrifuge 3415R | |
| ultracentrifuge tube | Beckman | 344058 | |
| PBS (Phosphate-buffered saline) | Corning | 46-013-CM | |
| BCA Protein Assay | Thermo Fisher Scientific | 23228 | |
| Transmission Electron Microscopy | Hitachi | HT7700 | |
| gelatin | STEMCELL | 7903 | |
| 10-cm culture dish | Falcon | 353003 | |
| 6-well culture dish | Corning | 3516 | |
| female C57BL/6 mice | NLAC (National Laboratory Animal Center | ||
| FBS (Fetal Bovine Serum) | BioWest | S01520 | |
| gentamycin | Thermo Fisher Scientific | 15710072 | |
| Pen/Strep | Corning | 30-002-Cl | |
| DNase I | 5PRIMER | 2500120 | |
| isofluorane | Halocarbon | NPC12164-002-25 | |
| formaldehyde | MACRON | H121-08 | |
| EtOH (Ethanol) | J.T. Baker | 800605 | |
| glacial acetic acid | Panreac | 131008.1611 | |
| aluminum potassium sulfate | Sigma-Aldrich | 12625 | |
| Xylene | Leica | 3803665 | |
| 0.22 μm membranes | Merck Millipore | Millex-GP | |
| AUTOCLIP Wound Clips, 9 mm | BD Biosciences | 427631 | |
| AUTOCLIP Wound Clip Applier | BD Biosciences | 427630 | |
| CellMask™ Deep Red | Thermo Fisher Scientific | C10046 | plasma membrane stain |
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Cite This Article
Lin, M., Chen, S., He, P., Luo, W., Li, H. Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes. J. Vis. Exp. (124), e55736, doi:10.3791/55736 (2017).