【摘要】 目的比较脐血CD34+细胞体外扩增前后生物学特征及HOXB4基因表达的变化。方法分离新鲜人脐血单个核细胞(MNC),应用免疫磁珠分选系统(MiniMACS)分选CD34+细胞,在含细胞因子组合FL+SCF+TPO+IL-3的IMDM+10%FBS培养基中体外培养2周,分别于培养第0、7、14天计数有核细胞(nucleatedcells,NC)数,流式细胞仪分析CD34+细胞比例,RT-PCR检测HOXB4基因的表达变化。并取第0天与第7天的细胞于甲基纤维素半固体培养基中培养14d,光镜下计数造血干/祖细胞集落形成数目。结果经MiniMACS分选,CD34+细胞比例由分选前的(1.1±0.25)%升至(83.17±8.67)%,CD34+细胞回收率为(72.90±8.75)%。体外培养第7天与第14天CD34+细胞的比例分别降至(4.8±0.64)%与(0.6±0.27)%(P<0.05);NC和CD34+细胞均有不同程度的增加,扩增7d与14d的NC数、CD34+细胞数分别较第0天扩增(76.2±14.9)倍、(254.0±48.85)倍与(3.9±0.69)倍、(1.8±0.64)倍,不同时间点差异有统计学意义(P<0.05)。经过14d半固体培养,扩增7d的造血干/祖细胞集落形成数目是未扩增的3.75倍(P<0.05)。RT-PCR检测HOXB4基因在第0天的CD34+细胞中高表达,但其表达量随着体外培养时间的延长而下降(P<0.05)。结论细胞因子组合FL+SCF+TPO+IL-3可有效扩增造血干/祖细胞,培养第7天较第14天的CD34+细胞扩增倍数高,但CD34+细胞表型在体外培养过程中迅速下降,与CD34+细胞自我更新能力相关的HOXB4基因的表达水平也随体外培养时间的延长而下降。更多还原

【Abstract】 Objective To compare the changes of biological characteristics and HOXB4 gene expression before and after expansion of human umbilical cord blood( HUCB) CD34+ cells in vitro. Methods CD34+ cells were separated from HUCB by MiniMACS and cultured in 10% FBS + IMDM including the cytokine combination of FL, SCF,TPO and IL-3. In the period,we observed cell morphology and biological properties,counted the number of nucleated cells ( NC) and colony-forming units( CFU) ,analysed CD34+ cells ratio with flow cytometry,and detected the HOXB4 gene expression changes by RT-PCR at different time points ( day 0,7,14 ) . Results The purity of selected CD34+ cell reached to ( 83. 17 ± 8. 67 ) % . The ratio of CD34+ cell decreased to ( 4. 78 ± 0. 64 ) % and ( 0. 82 ±0. 13) % on 7 days and 14 days culture. There was varying increase in the proportion of nucleated cells ( NC) and CD34+ cell,which the number of NC and CD34+ cell were respectively augmented( 76. 2 ± 14. 9) times, ( 254. 0 ±48. 85) times,( 3. 9 ± 0. 69) times and( 1. 8 ± 0. 64) times ( P < 0. 05) . The number of CFU on d7 was 3. 25 folds compared with day 0 culture. HOXB4 gene was highly expressed in separated CD34+ cells on day 0,but it declined rapidly in vitro expansion compared with its pre-culture level( P < 0. 05) . Conclusions The combination of FL + SCF + TPO + IL-3 enhances HUCB CD34+ cells effectively,and there is high amplification efficiency on 7 days culture compared with 14 days,but the self-renewal potential of CD34+ cells decrease in vitro culture.更多还原