小鼠骨髓来源肥大细胞的诱导培养及功能鉴定-东南大学学报医学版

小鼠骨髓来源肥大细胞的诱导培养及功能鉴定

单位：广州医科大学附属第二医院/呼吸疾病国家重点实验室变态反应研究室/变态反应国家临床重点专科/广东省过敏反应与免疫重点实验室, 广东广州 510260

分类号：Q813.11

出版年·卷·期（页码）：2015·34·第五期（684-688）

摘要：

目的:探讨体外诱导培养小鼠骨髓细胞来源肥大细胞的方法,为体外组胺激发试验提供试材。方法:采用白细胞介素3(IL-3)和干细胞因子(SCF)联合刺激小鼠骨髓细胞4、6周诱导其分化为肥大细胞,光镜下观察细胞形态,甲苯胺蓝染色观察细胞成熟度,蛋白免疫印迹法分析表面FcεRⅠ和CD117表达情况,HistaReader 501组胺仪检测细胞脱颗粒能力。结果:诱导培养4、6周后的细胞为大小均一且具折光性的圆形悬浮细胞;甲苯胺蓝染色显示培养4、6周后的细胞胞核染为蓝色,胞质含有丰富的粗大紫红色颗粒,镜下统计成熟肥大细胞比例达85%以上;蛋白免疫印迹法分析发现培养4、6周后的细胞皆表达FcεRⅠ和CD117,其中培养6周的细胞FcεRⅠ表达水平更高;组胺仪检测培养6周的细胞发现,经浓度为100、500、1 000 μmol·L^-1的钙离子载体刺激后,与阴性对照组相比,细胞组胺释放量依次为3.86%、17.04%及23.47%(P<0.05),其脱颗粒具有钙离子载体浓度依赖性。结论:获得大量高纯度的小鼠骨髓来源肥大细胞,可促进后期的组胺激发试验及Ⅰ型变态反应机制研究。

Objective: To investigate induced culture methods of bone marrow-derived mast cells(BMMCs) to provide materials for allergen challenge assay. Methods: BMMCs were differentiated from bone marrow cells cultured for 4 to 6 weeks with interleukin-3(IL-3) and stem cell factor (SCF) supplemented. The cell features were characterized by light microscopy and toluidine blue staining. The maturity of the cells was investigated by analyzing the membrane markers FcεRⅠand CD117 by Western blotting, and potency of BMMCs was calculated through histamine release assay by HistaReader 501 instrument. Results: The induced BMMCs, all in uniformed size with refraction, were observed to be filled with typical purple granules. The mature mast cells accounted for more than 85% of all cells. FcεRⅠand CD117 were positively expressed in BMMCs and FcεRⅠwere more expressive after 6 weeks of culture. The rate of histamine release by BMMCs cultured for 6 weeks were 3.86%, 17.04% and 23.47% (P<0.05) after stimulated with calcium ionophore at concentrations of 100, 500 and 1 000 μmol·L^-1, compared to control group. Conclusion: A large amount of highly purified BMMCs have been obtained, which will facilitate the subsequent allergen challenge assay and mechanism research on typeⅠallergy.

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