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目的:在体外利用小鼠骨髓干细胞诱导获得高纯度的肥大细胞。方法:从小鼠股骨获得骨髓细胞,放入含10%胎牛血清的RPMI1640培养基中,加入不同浓度的刺激因子,即IL-3和SCF各分别为10ng/ml、15ng/ml、20ng/ml,放入CO2培养箱中培养4周,用免疫组化观察CD117的表达,用流式细胞术检测CD117和FcεRIα双阳性细胞的比例,用甲苯胺蓝染色法观察细胞的成熟度,并与较常用的肥大细胞系P815做比较。结果:培养获得的肥大细胞,其中IL-3和SCF分别为10ng/ml和15ng/ml时均可在4周后得到纯度大于95%的CD117和FcεRIα双阳性肥大细胞,而20ng/ml时FcεRIα的表达却有所降低,双阳性细胞仅达90%,而P815细胞系表达FcεRIα的比例很低。免疫组化发现培养获得的细胞大部分均表达CD117。甲苯胺蓝染色发现培养获得的细胞核被染成深蓝色,胞浆中布满大量紫红色的颗粒,呈现肥大细胞的典型特征。而P815却少见典型的紫红色异染颗粒。结论:用IL-3和SCF各10ng/ml可在体外成功培养获得高纯度的小鼠骨髓源性肥大细胞,且其在表型和成熟度方面均好于P815细胞系。
OBJECTIVE: To induce high purity mast cells induced by mouse bone marrow stem cells in vitro. Methods: Bone marrow cells were obtained from the femur of mice and placed in RPMI1640 medium containing 10% fetal bovine serum. Different concentrations of stimulation factors (IL-3 and SCF) were added at 10ng / ml, 15ng / ml and 20ng / ml respectively The cells were cultured in CO2 incubator for 4 weeks. The expression of CD117 was observed by immunohistochemistry. The percentage of CD117 and FcεRIα double positive cells was detected by flow cytometry. The cell maturation was observed by toluidine blue staining. The commonly used mast cell line P815 is compared. Results: The mast cells obtained in this study showed that both CD117 and FcεRIα double positive mast cells with purity greater than 95% could be obtained in 4 weeks after IL-3 and SCF were 10ng / ml and 15ng / ml, respectively. However, But the expression of double positive cells was only 90%, while the percentage of FεRIα expression in P815 cell line was very low. Immunohistochemistry showed that the majority of cultured cells expressed CD117. Toluidine blue staining found that the nuclei obtained in culture were dyed dark blue, the cytoplasm was filled with a large number of purple particles, showing the typical characteristics of mast cells. The P815 rare rare purple exotic particles. CONCLUSION: High purity mouse bone marrow-derived mast cells can be successfully cultured in vitro with 10 ng / ml IL-3 and SCF, respectively, and are better than P815 cell line in terms of phenotype and maturity.