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三类骨髓基质细胞条件培养液体外扩增巨核系细胞

黄艳红, 王绮如

中南大学湘雅医学院血液生理研究室.湖南,长沙 410078

摘要

为了研究骨髓成纤维细胞、内皮细胞和巨噬细胞条件培养液对巨核细胞的体外扩增作用,该文通过分离纯化骨髓成纤维细胞、内皮细胞和巨噬细胞,收集无血清骨髓成纤维细胞条件培养液(fibroblast conditioned medium,F--CM)、骨髓内皮细胞条件培养液(endothelial cell conditioned medium,E--CM)和骨髓巨噬细胞条件培养液(macrophage conditioned medium,M--CM),分别离心、超滤,获得原液、分子量(MW)>10 kDa组分及MW<10 kDa组分。将三种无血清条件培养液及其>10 kDa组分和<10 kDa组分,分别加入巨核细胞体外液体培养体系,观察成熟巨核细胞的生成。结果表明:F--CM和E--CM及其>10 kDa组分加入培养体系后,成熟巨核细胞的生成显著增多,分别为对照组的(287.33±16.77)%、(141.21±17.47)%、(236.67±39.72)%和(179.03±30.98)%;F--CM的作用明显大于E--CM({sl P}<0.001);F--CM和E--CM的<10 kDa组分、M--CM及其>10 kDa组分和<10 kDa组分加入培养体系后成熟巨核细胞数无明显改变。分别将F--CM、E--CM、M--CM加入巨核细胞液体扩增体系,24 h后取细胞作巨核细胞集落形成单位(colony forming unit--megakaryocyte,CFU--Meg)的测定。结果表明:F--CM和E--CM对CFU--Meg生成均有促进作用,M--CM对CFU--Meg生成无明显影响,扩增强度分别为扩增前的(168.18±30.24)%、(215.17±17.4)%和(85.0±7.0)%。逆转录聚合酶链式反应(reverse transcription--polymerase chain reaction,RT--PCR)实验结果表明:骨髓成纤维细胞表达促血小板生成素(thrombopoietin,TPO),不表达转化生长因子#beta#1(transforming growth factor--#beta#1,TGF--#beta#1);骨髓内皮细胞表达TGF--#beta#1,不表达TPO。上述结果提示:F--CM、E--CM及其>10 kDa组分均对巨核系成熟细胞及巨核系祖细胞体外扩增有促进作用;F--CM对巨核系成熟细胞体外扩增的促进作用强于E--CM。

关键词： 骨髓; 基质细胞; 条件培养液; 巨核细胞; 扩增

Stimulation of bone marrow stromal cells conditioned medium on the expansion of mature megakaryocytes and colony forming unit--megakaryocyte {sl in vitro}

Huang Yanhong, Wang Qiru

Research Laboratory of Blood Physiology, Xiang Ya Medical School,Central South University.Changsha 410078,Hunan

Abstract

In this study, the effects of bone marrow stromal cells conditioned medium
on the expansion of mature megakaryocytes and CFU-Meg in vitro were investigated. The serum-free bone marrow fibroblast conditioned medium ( F-CM ), bone marrow endothelial cell conditioned medium ( E-CM ) and bone marrow macrophage conditioned medium ( M-CM ) were collected and ultrafiltrated by using a centriprep 10. F-CM, E-CM, M-CM, the retentate ( >10k F-CM, >10k E-CM and >10k M-CM ) contained substances whose molecular weight was more than 10k and the filtrate ( <10k F-CM, <10k E-CM and <10k M-CM ) contained substances whose molecular weight was less than 10k were added in liquid culture system respectively . The results showed that F-CM, >10k F-CM and E-CM, >10k E-CM significantly promoted the expansion of mature megakaryocytes in liquid culture system，the percentage of mature megakaryocytes compared with 0h control were （287.33±16.77）%，（236.67±39.72）%，（141.21±17.47）% and （179.03±30.98）%. But <10k F-CM, <10k E-CM, M-CM, >10k M-CM and <10k M-CM had no positive effects on the expansion of mature megakaryocytes. The effects of F-CM, E-CM or M-CM on the expansion of CFU-Meg were also investigated. The results indicated that F-CM and E-CM promoted the expansion of CFU-Meg in liquid culture system. M-CM had no positive effect on the expansion of CFU-Meg. the percentage of CFU-Meg compared with 0h control were （168.18±30.24）%，（215.17±17.4）% and（85±7）%.The results of reverse transcription-polymerase chain reaction ( RT-PCR ) showed that TGF-#beta#1 mRNA was expressed in bone marrow endothelial cells and was not expressed in bone marrow fibroblasts, TPO mRNA was expressed in bone marrow fibroblasts and was not expressed in bone marrow endothelial cells. These results suggest that F-CM, >10kDa F-CM and E-CM,>10kDa E-CM significantly promoted the expansion of mature megakaryocytes and CFU--Meg in liquid culture system. The effect of F-CM on the expansion of mature megakaryocytes is better than that of E-CM.

Key words: Bone marrow;stromal cells;Conditioned medium;megakaryocyte;expansion

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引用本文：

黄艳红, 王绮如. 三类骨髓基质细胞条件培养液体外扩增巨核系细胞[J]. 生理学报 2005; 57 (2): .

Huang Yanhong, Wang Qiru. Stimulation of bone marrow stromal cells conditioned medium on the expansion of mature megakaryocytes and colony forming unit--megakaryocyte {sl in vitro}. Acta Physiol Sin 2005; 57 (2): (in Chinese with English abstract).