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目的:探讨大鼠外周血中是否存在非黏附间充质干细胞(MSCs)以及1,25-(OH)2D3能否刺激它们形成成纤维细胞集落形成单位(CFU-f)。方法:从大鼠外周血中分离单核粒细胞(PBMGC)组份,在缺乏和添加10-8mol/L1,25-(OH)2D3条件下进行贴壁培养。培养至4天和8天时,将未贴壁细胞转移到新的培养皿中继续培养,即:“pour-off”培养,培养16天形成的细胞进行亚甲基蓝染色,计数CFU-f数目,并对4天“pour-off”培养形成的细胞进行免疫细胞化学染色。结果:PBMGC培养及4天和8天非黏附PBMGC培养都能形成CFU-fs,而1,25-(OH)2D3能够明显地促进它们形成CFU-f。非黏附细胞形成的克隆表达波形蛋白(vimentin)、α-平滑肌肌动蛋白(α-SMA)和Ⅲ型胶原,而不表达Ⅰ型胶原。结论:大鼠外周血中存在非黏附MSCs,在体外培养能够形成CFU-fs,而1,25-(OH)2D3具有促进外周血MSC扩增的作用。
OBJECTIVE: To investigate whether the presence of non-adherent MSCs and 1,25- (OH) 2D3 in peripheral blood of rats can stimulate their formation of fibroblast colony forming units (CFU-f). Methods: Mononuclear cells (PBMCs) were isolated from peripheral blood of rats and cultured in the absence and addition of 10-8mol / L, 25- (OH) 2D3. After 4 days and 8 days of culture, the non-adherent cells were transferred to a new culture dish to continue the culture, ie, “pour-off” culture, cultured cells formed for 16 days for methylene blue staining, counted the number of CFU-f, And immunocytochemically stained cells formed on a 4-day “pour-off” culture. Results: CFU-fs could be formed in both PBMGC culture and non-adherent PBMGC cultures on day 4 and day 8, while 1,25- (OH) 2D3 markedly promoted their formation of CFU-f. Clones formed by non-adherent cells expressed vimentin, alpha-SMA and type III collagen, but not type I collagen. CONCLUSION: Non-adherent MSCs exist in peripheral blood of rats, which can form CFU-fs in vitro. However, 1,25- (OH) 2D3 can promote MSC proliferation in peripheral blood.