小鼠骨髓源性肥大细胞的培养及鉴定  

Cultivation and Identification of Bone Marrow Mast Cells in Mouse

作　　者：张维娜[1] 吴轲[1] 周鸿敏[1] 彭清臻[2] 何文涛[1] 高英[1] 林星光[1] 方泽民[1] 陈忠华 

机构地区：[1]华中科技大学同济医学院附属同济医院,武汉430030 [2]孝感市中心医院

出　　处：《内科急危重症杂志》2009年第2期95-97,共3页Journal of Critical Care In Internal Medicine

基　　金：国家自然基金资助项目(No:30772038136071989);国家973规划项目(No:2009CB522407)

摘　　要：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 obtain pure mast cells from mouse bone marrow stem cells in vitro. Methods： Bone marrow cells were isolated from C57BL/6 mice. Then the cells were cultured with different concentration （10 ng/ ml, 15 ng/ml,20 ng/ml） of IL-3 and SCF. After four weeks cultivation, immunohistochemistry was used to detect the CD117 on the cells. Flow cytometry was used to identify the purity of double-positive （CD117 ＋ FcεRIα ＋ ） mast cells. Toluidine blue was used to detect the function of mast cells. P815 cells were tested by flow cytometry and toluidine blue. Results： After cultured for four weeks with 10ng/ml or 15ng/ml IL-3 and SCF, the purity of doublepositive cells was more than 95%. But the purity was only about 90% with 20ng/ml IL-3 and SCF. The percentage of double-positive cells was very low in P815 cells. The purple granules were found in bone marrow mast cells but not in P815 cells by toluidine blue. CDl17 was detected on almost all the bone marrow mast cells by immunohistochemistry. Conclusions： Pure mast cells can be obtained by cultivation with 10ng/ml IL-3 and SCF from mouse bone marrow cells. The cells are better than P815 cells in the phenotype and maturity.

关 键 词：肥大细胞 骨髓 小鼠 培养 

分 类 号：Q25[生物学—细胞生物学]