小鼠腹腔及骨髓源性肥大细胞的培养与鉴定

目的 探讨小鼠腹腔及骨髓源性高纯度肥大细胞体外诱导培养方法，并鉴定其功能。方法 分别从小鼠腹腔灌洗液及股骨获得腹腔及骨髓细胞，用白细胞介素3和干细胞因子分别联合诱导培养2周及4周，光镜下观察细胞形态，甲苯胺蓝染色鉴定肥大细胞成熟度，流式细胞仪检测细胞表面分子CD117和FcεRⅠα的表达情况，不同浓度Compound48/80刺激后镜下计数肥大细胞脱颗粒率，分光光度法测定肥大细胞β己糖胺酶释放率。结果 分别诱导培养2周及4周后，小鼠腹腔及骨髓细胞呈大小均一且具折光性的悬浮细胞。甲苯胺蓝染色示上述两种细胞胞质内含紫红色的异染颗粒。两种细胞表面CD117及FcεRⅠα单阳性率均高于95%，双阳性率分别为97.68% ± 0.80%及96.12% ± 0.76%。100和1 000 mg/L Compound48/80刺激组与空白对照组肥大细胞相比，脱颗粒率差异有统计学意义（均P < 0.01）。100 mg/L Compound48/80组骨髓源性肥大细胞以及10 mg/L、100 mg/L Compound48/80组腹腔源性肥大细胞β己糖胺酶释放率均较空白对照组显著升高（P < 0.01或0.05）。结论 白细胞介素3和干细胞因子联合可诱导小鼠骨髓干细胞及腹腔细胞定向分化增殖，从而获得高纯度成熟且具有脱颗粒功能的肥大细胞，为后续细胞生物学研究奠定基础。

Cultivation and identification of mouse peritoneal and bone marrow-derived mast cells

Shao Yixin, Wang Duoqin, Shen Yanyun, Zhu Yiqi, Xu Jinhua, Tang HuiDepartment of Dermatology, Zhongshan Hospital, Fudan University, Shanghai 200040, ChinaCorresponding author: Tang Hui, Email: tanghuihuashan@163.com【Abstract】 Objective To explore the in vitro culture methods for oriented differentiation of peritoneal cells and bone marrow cells into high-purity mast cells, and to identify the function of these mast cells. Methods Peritoneal cells and bone marrow cells were isolated from the peritoneal cavity lavages and femur of C57BL/6 mice, and cultured with both interleukin-3 （IL-3） and stem cell factor for 2 and 4 weeks respectively. Light microscopy was performed to observe the morphology of these cells, toluidine blue staining to identify the degree of maturity of these mast cells, and flow cytometry to measure the of cell surface markers CD117 and FcεRⅠα. After the stimulation with compound 48/80 at different concentrations, the degranulation rate of mast cells was counted under the microscope, and β-hexosaminidase release rate was measured by spectrophotometry. Results After 2- or 4-week culture, the mouse peritoneal and bone marrow cells all manifested as refractive suspension cells of uniform size. Toluidine blue staining showed violaceous metachromatic granules in the cytoplasm of the two kinds of cells. The proportions of CD117 or FcεRⅠα single-positive peritoneal and bone marrow-derived mast cells were all more than 95%, and the proportions of CD117/FcεRⅠα double-positive peritoneal and bone marrow-derived mast cells were 97.68% ± 0.80% and 96.12% ± 0.76% respectively. The degranulation rates of mast cells in the 100- and 1 000-mg/L compound 48/80 groups significantly differed from those in the blank control group （all P < 0.01）. Compared with the blank control group, the β-hexosaminidase release rates significantly increased in bone marrow-derived mast cells in the 100-mg/L compound 48/80 group and peritoneal mast cells in the 10- and 100-mg/L compound 48/80 groups （P < 0.01 or 0.05）. Conclusion IL-3 and stem cell factor can co-induce the directed differentiation and proliferation of mouse bone marrow stem cells and peritoneal cells, so as to harvest high-purity mature degranulated mast cells, and lay a foundation for subsequent cell biology research.