$ j' I/ F* e0 M2 u3 s1 E 【摘要】 为了探讨胚胎骨髓基质细胞(FBMSC)联合细胞因子对脐血单个核细胞(MNC)中CD133 细胞的体外扩增作用,将新鲜脐血(CB)中分离出来的MNC接种于无血清培养体系中培养14天。实验分为4组:C组为空白对照组,不含基质细胞和细胞因子;S组为单用基质细胞组;F组为单用细胞因子组;SF组为联合使用基质细胞和细胞因子组。在第0,6,10及14天检测有核细胞总数、CD133 细胞数及集落形成单位(CFU)数。结果表明:各时间点SF组有核细胞总数的扩增倍数均高于其它组;除了第14天外,SF组在第6、10天时CD133 细胞数、CFU数的扩增倍数均高于其它组。结论: 胚胎骨髓基质细胞对延缓造血细胞的分化具有重要的作用,基质细胞联合细胞因子可以有效的扩增脐血单个核细胞及其中的CD133 细胞,这是一种比较接近于临床移植要求的造血细胞体外扩增方法。 9 l: W* Y+ T9 s% u2 N 【关键词】骨髓基质细胞; 造血干细胞; 细胞扩增; CD133 细胞; 脐血; 细胞因子( q& }5 n, F; m( r
In Vitro Expansion of Cord Blood CD133 Cells Supported by Bone Marrow Stromal Cells and Cytokines0 Z" h4 z K* Z; {& X
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MAO Ping, ZENG JingLong, WANG CaiXia, DU QingHua, q2 F' U q+ X+ l7 `
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Department of Hematology, The First Guangzhou Municipal People Hospital Affiliated to Guangzhou Medical College, Guangzhou 510182, China: V! M7 K% f' m& K( _0 h+ q1 b% Q# B
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AbstractThe aim of this study was to investigate the effects of human fetal bone marrow stromal cells (FBMSC) in combination with exogenous cytokines on supportingin vitro expansion of CD133 cells in cord blood mononuclear cells (MNC). MNCs separated from cord blood (CB) were cultured for up to 14 days in a serumfree system with FBMSC or exogenous cytokines or both of them. On day 0,6,10 and 14, total nucleated cells (TNC) were counted;CD133 cells were quantifiedby FACS, and hematopoietic progenitor cells were assessed by semisolid culture assay. The results showed that the number of TNC was remarkably increased in FBMSC and cytokine group, the expansion of CD133 cells andCFU were increased in FBMSCand cytokine group except that on day 14. It is concluded that FBMSC play an important role in delaying the differentiation of hematopoietic cells. FBMSC in combination with exogenous cytokines can promote the effective expansion of CB MNC and CD133 cells, this expanding system may meet the needs for clinical application of expanded CD133 cells.8 h6 ]; R i- u0 h4 o
" g: K& X% m/ {: L* t目前许多研究者对HSC扩增的研究都是使用分离纯化的CD34 细胞或者CD133 细胞。对细胞分选之后进行培养,虽然能够获得较大的扩增倍数,而且扩增效果也较稳定[13],但CD34 细胞能由CD133 CD34-细胞体外培养产生[14],而CD133 细胞也可能经由CD133-细胞产生[15]。因此分选细胞不可避免地造成CD34 细胞或者CD133 细胞的丢失,同时也可能丢失比它们更加早期的造血细胞。而用于移植的脐血主要是以单个核细胞或全血的方式来储存的,脐血造血干细胞移植的植入速度也主要和整个有核细胞数有关[16]。所以,本实验选用脐血MNC作为培养的起始细胞。' w* g; o i0 z- ?1 H" B( e1 l# h
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我们的研究发现,单用SCF TPO FL细胞因子组合培养脐血MNC时,虽然CD133 细胞能得到一定量的扩增,但有核细胞总数都一直呈下降的趋势。其原因可能是我们所用的细胞因子都是一些早期作用因子,在扩增早期HSC的同时,较晚期阶段的造血细胞很快就衰老死亡。CFU和CD133 细胞检测的数据也表明,单用细胞因子也造成了早期HSC的迅速耗竭。 1 B. T7 R" u/ f6 Z% @5 s$ R; L. [; F# }% g0 u% X! w5 F
在单用基质细胞的实验组中,有核细胞总数和CD133 细胞都有一定的扩增,但增幅较小。初期6天有核细胞总数有所下降,但同时期CD133 细胞则是有所扩增。推测是因为CD133 早期造血细胞主要是处于G0/G1期[17],细胞进入增殖周期需要一定的时间,而这一时期的晚期阶段造血细胞衰老死亡较快。经过14天的培养后,单用基质细胞组的CFU计数一直上升为第0天值的17.46±3.42倍,体现出基质细胞对延缓造血细胞的分化的重要作用。 / Y" l" r# p0 h; l; Z/ M9 Z3 @ G . u' l( y6 F0 o2 n; S, Z, S4 a5 h! W联合使用基质细胞和细胞因子时,有核细胞总数得到有效扩增的同时,CD133 细胞也得到了显著的扩增,比单用基质细胞或者单用造血因子都有明显的优势。在有核细胞总数的扩增方面,单用基质细胞总的趋势是扩增,单用细胞因子时是一直在下降,联合使用基质细胞和细胞因子组合时,则呈增加的趋势,这说明两者合用具有协同促进造血细胞增殖的效果。在进行造血干细胞移植时,我们需要的不仅是能长期造血的早期的造血干细胞,而且需要有助于短期内迅速恢复造血的处于较晚期阶段的造血祖细胞[18]。所以,基质细胞联合细胞因子可能是比较接近临床使用要求的扩增脐血的方法。 " G9 U, m% h& R% t 4 |& p7 o7 R& d单份脐血的容量及所含的HSC有限,不能够满足体重较大的儿童和成人患者的移植需要,对造血细胞进行体外扩增是有望解决这个问题的方法之一。Jaroscak等[19]用封闭的持续灌注的细胞培养系统扩增脐血造血细胞后进行Ⅰ期临床试验,虽然移植体外扩增后的细胞并不改变红系、粒系和血小板的植入时间,但观察期间输注的扩增后的脐血细胞对患者是安全、无毒性的。因此深入研究造血细胞增殖和分化的调控因素,改进细胞扩增的体系和方法,对脐血HSC移植有重要的意义。 % q* y" ~/ `9 j) X 【参考文献】3 o- o; A$ P1 G, y/ h( s9 b1 H
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}) x. A1 B' J+ T: I 19Jaroscak J, Goltry K, Smith A, et al. Augmentation of umbilical cord blood (UCB) transplantation with ex vivoexpanded UCB cells: results of a phase 1 trial using the AastromReplicell System. Blood, 2003; 101:5061-5067作者: 大小年 时间: 2015-6-4 16:59