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为了探索一种较理想的人骨髓巨核系祖细胞CFU-M的体外半固体培养方法,本文对血浆凝块法和甲基纤维素法进行比较,证实血浆凝块法培养的CFU-M开始出现于第5天,集落高峰在第10～12天,消失于第16天。甲基纤维素法CFU-M开始出现于第5～6天,集落高峰在第14～16天,集落消失在20天以后。前者集落呈弥散状或紧密状不一,可原位固定染色计数,标本易保存;后者集落呈紧密状,不能原位固定,可在倒置镜下根据细胞形状计数,或吸出细胞涂片染色,培养皿不能保存。当两种培养体系中加入相同的条件培养基时,CFU-M数无显著性差别。本文还介绍一种改良的血浆凝块法,该方法以PHA-LCM为条件培养基,并用抗血小板糖蛋白单抗和ABC染色法来鉴别生长的巨核细胞。此法集落产率高,鉴别指标客观特异,不需要荧光显微镜。14例正常骨髓标本的CFU-M的产率为136.4～42.5/5×10_5((?)±SD)。当培养体系中加入不同的条件培养基CFU-M的产率不同,其中以PHA-TCM所得CFU-M数最高。本文结果提示:用PHA-TCM作条件培养基的血浆凝块法结合用抗血小板GPⅡa单抗的ABC染色,是研究CFU-M体外增殖分化和探讨血小板性疾病发病机制的理想方法。
In order to explore a more ideal semi-solid culture method of human bone marrow megakaryocyte progenitor cells CFU-M in vitro, we compared the plasma clot method and methylcellulose method to confirm that CFU-M cultured in plasma clotting method began to appear On the fifth day, the peak of the colony disappeared on the 16th day on the 10th to 12th days. Methylcellulose CFU-M began to appear on the 5th to 6th day, the colony peak on the 14th to 16th day, and the colony disappeared after 20 days. The former colonies were diffuse or closely spaced, can be fixed in situ dyeing count, the specimen is easy to save; the latter was compact, can not be fixed in situ, under the inverted microscope according to the cell shape count, or suck smear cells , Petri dish can not be saved. When the same conditioned medium was added to both culture systems, there was no significant difference in the number of CFU-M. This article also describes an improved plasma clot method that uses PHA-LCM as a conditioned medium and identifies the growing megakaryocytes using anti-platelet glycoprotein monoclonal antibody and ABC staining. This method of colony yield, objective identification of objective specificity, does not require fluorescence microscopy. The yield of CFU-M from 14 normal bone marrow specimens was 136.4 ~ 42.5 / 5 × 10_5 ((±) ± SD). The yield of CFU-M with different conditions was different in the culture system, among which the highest number of CFU-M was obtained with PHA-TCM. Our results suggest that plasma clotting with PHA-TCM as medium and ABC staining with anti-platelet GPIIa monoclonal antibody are the ideal methods to study the proliferation and differentiation of CFU-M in vitro and explore the pathogenesis of platelet diseases.