白血病干细胞和骨髓基质细胞共培养的方法学探讨

目的 探讨白血病干细胞和骨髓基质细胞共培养的可行方法及扩增白血病干细胞的特点.方法 体外用两种方法共培养急性髓系白血病（AML）患者的骨髓单个核细胞（MNCs）：一种方法是将MNCs中的贴壁基质细胞单独培养,第2代或第3代基质细胞用丝裂霉素处理后做饲养层,再同悬浮造血细胞共培养;另一种方法是将MNCs持续培养即原代共培养.观察MNCs形态学特征、长期存活情况及增殖特性,用流式细胞仪检测分析细胞表面标志.结果 两种方法共培养的细胞部分可以长期存活,在共培养中有卵石样区域细胞及悬浮细胞团的形成.原代共培养的细胞所形成的卵石样区域数目多,排列规律.结论 两种方法都可以起到扩增白血病干细胞的作用.同经过丝裂霉素处理之后的饲养层相比,没有经过处理的骨髓基质细胞在共培养时能够更有效地扩增白血病干细胞.

Methodology research of co-culturing leukemia stem cells and bone marrow stromal cells

Objective To investigate the feasible methods of co-culturing the leukemia stem cells and bone marrow stromal cells and the characteristics of amplified leukemia stem cells. Methods Two methods were used to co-culture the bone marrow mononuclear cells （MNCs） of patients with acute myeloid leukemia （AML）. One method was co-culturing the suspended MNCs with feeder layer which were 2nd or 3ra generation stromal cells treated with mitomycin; another method was co-culturing the MNCs continually, which was also called primary co-culture. And then the morphology, long-term survival and proliferation of MNCs were observed, the MNCs＇ surface markers was analyzed by flow eytometry. Results Part of the MNCs could be long-time survival in both co-culture methods and there were pebble-like areas and suspended cell mass formation, especially in the primary co-culture. Conclusion Both co-culture methods can promote amplification of leukemia stem ceils. Compared with the mitomycin treated feeder layer, the primary co-culture method can better amplify the leukemia stem cells.