| 彭 霞,梁玉婷,林 堃,尹 悦,李 莉.小鼠骨髓来源肥大细胞的培养及鉴定[J].,2017,17(30):5807-5811 |
| 小鼠骨髓来源肥大细胞的培养及鉴定 |
| Culture and Identification of Bone Marrow-derived Mast Cells in Mice |
| 投稿时间：2017-04-22 修订日期：2017-05-18 |
| DOI：10.13241/j.cnki.pmb.2017.30.002 |
| 中文关键词: 小鼠 骨髓 肥大细胞 培养 β-己糖苷酶释放 |
| 英文关键词: Mouse Bone marrow Mast cell Culture β-hexosaminidase release |
| 基金项目:国家自然科学基金项目（81471593, 81601395）；上海市青年扬帆计划（16YF1409200） |
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| 中文摘要: |
| 摘要 目的：探讨小鼠骨髓来源肥大细胞体外大量培养及鉴定方法。方法：采用IL-3（10 ng/mL）和不同浓度的SCF联合刺激小鼠骨髓细胞，培养四周，采用普通光学显微镜观察细胞形态，牛鲍氏血细胞计数板进行细胞计数，甲苯胺蓝染色观察其胞浆颗粒物质，流式细胞仪检测细胞表面CD117和FcεRI的表达情况，β-己糖苷酶释放试验评估肥大细胞活化能力。结果：培养条件为10 ng/mL IL-3和20 ng/mL SCF时，悬浮细胞数目能达到1×108细胞，其中CD117和FcεRI双阳性细胞比例达90 %以上；甲苯胺蓝染色可见细胞胞浆含有大量紫红色颗粒；细胞经IgE-DNP致敏及DNP-HSA激发后，β-己糖苷酶释放率明显增加，且SCF能促进肥大细胞释放β-己糖苷酶。结论：采用10 ng/mL IL-3和20 ng/mL SCF诱导骨髓细胞能够获得大量高纯度的小鼠骨髓来源肥大细胞，为肥大细胞特性及功能学研究提供了基础。 |
| 英文摘要: |
| ABSTRACT Objective: To explore a method to obtain mass mast cells with high purity in vitro. Methods: Murine bone marrow cells were cultured in the medium containing 10 ng/mL IL-3 and different concentration of SCF in vitro. After 4 weeks, the morphology of cells was observed using optical microscopy, and the cell numbers were counted using NiuBao's blood count plate. Then, the particles in the cytoplasm of the cells were analyzed using toluidine blue staining. Besides, the expression of CD117 and FcεRI on the cell surface was detected by flow cytometry. Finally, the cell activation was evaluated by β-hexosaminidase release assay. Results: When cultured in the medium containing 10 ng/mL IL-3 and 20 ng/mL SCF, 1×108 suspension cells were obtained, and the ration of CD117 and FcεRI double positive cells was above 90%. A large number of purple red granules were observed in the cytoplasm of the cells. After sensitized with anti-DNP IgE and challenged with DNP-HSA, the rate of released β-hexosaminidase was significantly increased compare with control group, and β-hexosaminidase release of mast cells could be potentiated by SCF. Conclusion: A large number of mast cells with high purity could be obtained by stimulating the bone marrow cells by 10 ng/mL IL-3 and 20 ng/mL SCF, which may provide foundation for the characteristics and functional study of mast cell. |
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