摘要
目的探讨骨髓间充质干细胞(MSC)诱导的成骨细胞复合人源生物衍生骨,构建三维培养体系,观察模拟的造血龛(hematopietic niche)对脐血CD34+造血干／祖细胞(HSPC)增殖的支持作用。方法分选人骨髓MSC及胎儿颅骨成骨细胞,将培养3代的MSC定向诱导为成骨细胞,分别将①MSC、②颅骨成骨细胞、③MSC与成骨细胞混合物(比例为1:2)及④MSC诱导的成骨细胞作为饲养细胞复合在人源生物衍生骨上,构建三维培养体系,同时用相应的饲养细胞层构建对应的二维培养体系。免疫磁珠分选脐血CD34+细胞,接种于各培养体系,无外源性细胞因子条件下进行培养。显微镜下观察各饲养细胞与人源生物衍生骨复合情况,通过流式细胞术、半固体细胞集落培养观察比较各培养体系支持HSPC的增殖能力。结果倒置相差显微镜、扫描电子显微镜以及免疫荧光标记观察结果显示,已成功地将MSC、颅骨成骨细胞、MSC和成骨细胞混合物及MSC诱导的成骨细胞复合在人源生物衍生骨上,构建了造血干细胞三维培养体系。在无外源细胞因子作用下,脐血HSPC培养2周造血祖细胞集落(CFU-C)与长期培养起始细胞(LTC-IC)分析,三维培养体系各组(①～④)CFU-C明显多于相应二维培养体系各组(①681．00±44．43比438．20±64．24;②729．80±37．72比536．60±35．31;③697．80±54．09比464．80±41．53;④740．20±47．66比513．40±65．56),P<0．05,但三维培养体系各组间差异无统计学意义(P>0．05)。三维培养体系各组LTC-IC也明显高于相应二维培养体系(分别为①68．00±5．11比26．00±3．73;②93．00±6．12比38．60±4．93;③83．40±5．85比36．20±4．52;④126．00±11．89比59．40±4．25),P<0．05,且三维培养体系中MSC诱导的成骨细胞组(④)LTC-IC明显多于其他各组。脐血HSPC培养5周,二维培养体系中饲养细胞层已逐渐崩解、脱落、死亡;而三维培养体系中饲养细胞层生长情况较好。三维体系各组CFU-C集落分析显示,MSC诱导的成骨细胞组(④)与其他三组间差异有统计学意义(①112．00±14．21;②186．17±24．80;③126．67±11．52;④257．00±28．78),P<0．05。结论骨髓MSC诱导分化的成骨细胞复合人源生物衍生骨构建的三维培养体系,对于脐血HSPC体外培养具有类似体内造血龛的支持作用,成骨细胞对造血干／祖细胞生存、增殖具有重要的调控作用。
Objective To construct a model that imitate human hematopoietic niche with osteoblasts induced from human marrow mesenchymal stem cells（MSCs） and bio-derived tissue engineering bone, and to investigate the ability of the three-dimensional model to culture CD34^＋ umbilical cord blood cells ex vivo. Methods MSCs were isolated from adult human bone marrow and cultured, the third generation of MSCs were induced into osteoblasts. The osteoblasts isolated from skull were cultured. The four kinds of three-dimensional culture systems had been constituted by adhering to tissue engineering bone with ①human MSCs, ②osteoblasts of skull, ③MSCs/osteoblasts of skull（ 1： 2）, and ④osteoblasts induced from MSCs, respectively. As control, four two-dimensional culture systems were formulated by using those homologous cells as feeder cell layer. CD34^＋ cells were isolated from fresh umbilical cord blood samples with immunomagnetic beads and seeded into the models to culture ex vivo without exogenous cytokines. The combinations of feeder cells with tissue engineering bone were observed, and the abilities of either three or two dimensional models to culture HSCs/HPCs were evaluated by flow cytometry analysis, colony-forming assays, and long-term cultures assays. Results The four kinds of feeder cells were combined with tissue engineering bone successfully, which were identified by inverted microscope, electron microscope, and immunofluorescence staining. At two weeks culture for HSCs/HPCs without exogenous cytokines, the number of CFU-C in three-dimensional groups were obviously more than those in two-dimensional groups （①681. 00 ± 44. 43 vs 438. 20 ± 64. 24, ②729.80 ± 37.72 vs 536.60 ± 35.31, ④697.80 ± 54.09 vs 464.80 ± 41.53, 740.20 ± 47.66 vs 513.40 ± 65.56, P 〈 0.05 ）, but there was no difference in each three-dimensional groups（ P 〉 0.05）. The number of LTC-IC in three-dimensional groups were also more than those in two-dimensional groups（①68.00 ± 5.11 vs 26.00 ± 3.73, ②93.00 ± 6.12 vs 38.60 ± 4.93, ③83.40 ± 5.85 vs 36.20 ± 4.52,④126.00 ± 11.89 vs 59.40 ± 4.25, P 〈 0.05 ） , and was evidently higher in osteoblasts induced from MSCs group than that in other groups（P 〈 0.05）. At five weeks eulture, the feeder eell layer in two-dimensional eulture systems showed cells disaggregating, ablating, and dying, but that in three-dimensional eulture systems grew well. The number of CFU-C in osteoblasts indueed from MSCs group was obviously higher than that in other groups （112.00±14.21, 186.17±24.80, 126.67±11.52, 257.00±28.78, P〈0.05）. Conclusions Athreedimensional model with osteoblasts indueed from human MSCs and bio-derived tissue engineering bone were constituted , whieh could imitate human haematopoietic niehe and support HSCs/HPCs of umbilieal eord blood in long-term eultures, and it also proved that osteoblasts play a erueial role in HSCs/HPCs regulation.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2006年第12期795-800,共6页
Chinese Journal of Hematology
关键词
成骨细胞
造血龛
骨髓间充质干细胞
生物衍生骨
Osteoblasts
Hematopoietie niehe
Mesenehymal stem eells
Bio-derived tissue engineering bone
