小鼠骨髓巨核系祖细胞集落(CFU-MK)培养方法的建立及染色方法的改进

Establishment of a cultivation method for murine bone marrow colony forming units-megakaryocyte (CFU-MK) and improvement of staining method

目的 :为深入探讨巨核系祖细胞增殖、分化的调控机制提供有效的技术途径。方法 :建立了小鼠骨髓CFU_MK的培养方法 ,并对影响集落形成及染色等条件进行了改进。结果与结论 :单因子rhIL_3,rhIL_6 ,rhGM_CSF ,rhEPO ,rhTPO和脾条件培养液及再障狗血清均具有不同程度的刺激CFU_MK生成的作用 ,尤以再障狗血清的刺激活性最强 ;接种细胞数为 (2～ 3)× 10 5 ml,培养 7～ 10d时CFU_MK的集落形成率较佳 ;采用改进后的不转移、不洗涤、原位复染并 4℃放置 2 4h的染色条件 。

Objective:To provide a technical method for studying the proliferation and differentiation mechanism of megakaryoblast.Methods:Cultivation method for mouse bone marrow CFU_MK was established and the staining method was improved for getting better effects.Results and Conclusions:Various cytokines such as rhIL_3, rhIl_6, rhGM_CSF, rhEPO, rhTPO, and the conditional culture medium for spleen as well as aplastic anemic dog serum used separately could stimulate the formation of CFU_MK to different extent, especially the aplastic anemic dog serum had the strongest stimulating action on colony formation. The formation rate of the CFU_MK was highest under the condition of inoculation of (2-3)×10 5 cells/ml and 7-10 days of cultivation. The higher rate of staining CFU_MK could be acquired using the method of no transfer,no washing,re_staining in situ and keeping at 4℃ for 24*!h, as compared with the conventional method.