微载体支持体外造血研究

Research on Ex Vivo Hematopoiesis Supported by Microcarriers

为了探索体外维持造血干细胞自我更新潜能 ,抑制过度分化以及大量扩增造血细胞的技术 ,我们将小鼠骨髓基质细胞贴附于微载体上 ,再接种小鼠骨髓造血细胞共同培养 (G2组 )。在此基础上 ,用海藻酸钠包埋微载体基质细胞和造血细胞制成凝胶珠进行培养 (G1组 ) ;设置单纯微载体基质细胞 (G3组 )和单纯骨髓细胞 (G4组 )培养作为对照。对比观察 ,显微摄影 ,计数细胞总数、CFU GM集落数和CD34阳性细胞百分率。培养 2周观察到造血细胞粘附在微载体基质细胞上或龛在微载体基质细胞间并形成造血灶。 3次实验结果显示 ,G2组CFU GM集落总数显著高于G4组 ,同时也显著高于G3加G4组集落总数之和 (t=6.5 5 3和t =5 .4 94 ;P <0 .0 5 ) ;G2组CD34+ 细胞百分率高于其他各组 ,与CFU GM测定结果趋向一致。实验表明 ,微载体基质细胞有较好的支持体外造血的效果 ,至少可维持 4周 。

To explore methods of maintaining the self-renewal capacity of hematopoietic stem cells, inhibiting their overdue differentiation and expanding hematopoietic cells massively, the murine bone marrow stromal cells were coated on microcarriers, then co-cultured with hematopoietic cells from murine bone marrow as group 2 (G2). The G2 contents were wrapped by sodium alginate, then cultivated as group 1(G1). The only microcarriers coated with stromal cells as group 3 (G3) and the only bone marrow cells as group 4 (G4) were cultivated as control groups. Contrasting observation and microphotograph were performed; the number of total marrow cells, the colony efficiency of CFU-GM and the percentages of CD34 + cells were determined. Three repeated experiments indicated that the colony efficiency of CFU-GM before culture (G0) were 118.8±38.1/10 5 marrow cells, and the total outputs of CFU-GM (G0) were 9 501.3±3 049.0. After culture for two weeks, hematopoietic cells were adhered to or embedded in stromal cells coating the microcarriers, and had formed hematopoietic focus. The colony efficiency of CFU-GM per 10 5 mononuclear cells in group G1, G2, G3 and G4 averaged 30.9±13.7, 147.3±66.0, 23.4±23.1 and 15.9±8.1, respectively; the total outputs of CFU-GM in group G1, G2, G3 and G4 averaged 273.8±75.3, 9 424.8±7 933.7, 419.1±305.6 and 140.7±20.7, respectively; the measured CFU-GM output in group G2 was significantly higher than that in group G4, and still significantly higher than the sum of groups G3 and G4 (t=6.553, t=5.494; P<0.05). The percentage of CD34 cells before culture was 10.0±1.0; after cultuer for two weeks, the percentages of CD34 + cells in G1, G2, G3 and G4 averaged 4.0±1.0, 11.0±1.0, 3.3±1.5 and 2.2±0.8, respectively. The percentage of CD34 positive control (3T3 cells) was 17.0±1.0. This result was consistent with the result of CFU-GM outputs measured. These data suggest that microcarriers coated with stromal cells can perfectly support the ex vivo hematopoiesis at least to four weeks, while hematopoietic cells fixed by alginate are not significantly different from control groups. The hematopoiesis-simulating model of microcarriers is successful, whereas the hematopoiesis-simulating model of alginate macrocarriers can not support the ex vivo hematopoiesis.