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在模拟骨髓造血壁龛(hematopoietic niche)的氧分压条件下,探讨微囊化成骨细胞(osteoblasts,OB)对脐血造血干/祖细胞(HSPC)体外扩增的支持和调控机理.分离培养人髂骨OB,采用聚电解质络合法将第3代的OB以密度为8×105ml包埋在直径为0.5mm的明胶-海藻酸钠-壳聚糖(GAC)微胶珠中.将微珠+造血干/祖细胞(A′组)、造血干/祖细胞(B′组)及微珠(C′组)置于6孔板,在5%氧分压下进行培养.同时在20%常氧条件下设置同样分组培养作为对照(A,B,C).通过流式细胞分析和半固体细胞集落培养,观察比较各培养体系中造血干/祖细胞的扩增,并检测体系内白血病抑制因子(LIF)和白介素-6(IL-6)的含量变化以探讨作用机理.经过倒置相差显微镜观察,人成骨细胞在微珠中分散均匀,生长状态良好.微珠内部有丰富的孔道供营养物质传递,有大量造血干/祖细胞弱黏附于微珠表面.经过7天的培养,A′、B′、A、B四组造血细胞的扩增倍数分别为(49.0±4.6),(3.3±0.5),(17.7±1.2)和(1.9±0.2).A′、B′、A组的CD34+细胞分别扩增了(87.6±8.3),(2.2±0.3)和(14.9±1.0)倍,B组则出现下降.A′、B′、A、B四组CFU-Cs集落扩增倍数分别为(9.8±0.8),(3.5±0.4),(6.9±0.7)和(2.6±0.2).低氧共培养体系比常氧共培养体系和非共培养体系对造血干/祖细胞的扩增有更大的促进作用.A′、B′、C′中IL-6和LIF含量明显高于对应的A、B、C组,与扩增倍数的差异相对应.微囊化成骨细胞对造血干/祖细胞扩增有明显的促进作用,5%氧分压接近体内造血壁龛氧环境,在此环境中成骨细胞分泌细胞因子量增多并通过其对造血干/祖细胞的扩增进行调节.
To investigate the support and regulation mechanism of microencapsulated osteoblasts (OB) on in vitro expansion of cord blood hematopoietic stem / progenitor cells (HSPCs) under simulated oxygen partial pressure in hematopoietic niche. Human ilium OB was implanted into the gelatin-sodium alginate-chitosan (GAC) microbeads with a diameter of 0.5 mm at a density of 8 × 10 5 ml using polyelectrolyte complexing method. Beads + hematopoietic stem / progenitor cells (A ’group), hematopoietic stem / progenitor cells (B’ group) and beads (C ’group) were placed in 6-well plates and cultured at 5% The same grouping cultures were set as controls (A, B, C) under normoxic conditions.Flow cytometry analysis and semi-solid colony culture were used to observe the proliferation of hematopoietic stem / progenitor cells in each culture system (LIF) and interleukin-6 (IL-6) contents in human umbilical cord blood to explore the mechanism of action.Observed by inverted phase contrast microscopy, human osteoblasts were well dispersed in microbeads and grew well.The microbeads were rich in Channel for nutrient delivery, a large number of hematopoietic stem / progenitor cells weakly adhered to the surface of the beads after 7 days of culture, A ’, B’, A, B four (49.0 ± 4.6), (3.3 ± 0.5), (17.7 ± 1.2) and (1.9 ± 0.2), respectively.The CD34 + cells in group A ’, B’ ± 8.3), (2.2 ± 0.3) and (14.9 ± 1.0) times, respectively, but decreased in group B. The multiplication of CFU-Cs colonies in groups A, B, A and B were (9.8 ± 0.8) 3.5 ± 0.4), (6.9 ± 0.7) and (2.6 ± 0.2), respectively.The hypoxia co-culture system promoted the expansion of hematopoietic stem / progenitor cells more than nocturnal co-culture system and non-co-culture system.A The content of IL-6 and LIF in ’B’ and C ’was significantly higher than that of corresponding groups A, B and C, which corresponded with the difference of amplification fold.The microencapsulated osteoblasts had obvious amplification of hematopoietic stem / progenitor cells The 5% partial pressure of oxygen approaches the oxygen environment of the hematopoietic niche in the body where the amount of cytokines secreted by the osteoblasts is increased and the expansion of hematopoietic stem / progenitor cells is regulated by this.