动员人外周血巨核祖细胞体外扩增的研究_临床医学论文.docVIP

动员人外周血巨核祖细胞体外扩增的研究_临床医学论文 动员人外周血巨核祖细胞体外扩增的研究_临床医学论文 作者：夏婷，方建培，吴燕峰，徐宏贵，魏菁 【摘要】 本研究探讨多种细胞因子（TPO、SCF、FL、IL-1、IL-3、IL-6）组合的几种培养体系对人外周血CD34+细胞体外诱导扩增生成巨核细胞的作用，研究人外周血来源的巨核细胞体外扩增的最佳细胞因子组合及培养时间。用Ficoll-Hapaque分离法分离动员的外周血（MPB）单个核细胞，免疫磁珠法分离纯化CD34+细胞，并将其在含胎牛血清的液体培养体系中、各组细胞因子诱导下培养15天。在不同时间点采用血细胞计数板进行细胞计数，采用流式细胞术检测培养体系中CD41+细胞的含量；同时采用甲基纤维素半固体培养法进行巨核细胞集落培养，测定巨核细胞集落形成单位（CFU-MK）的数量。结果表明：经过15天的培养，在MPB来源的CD34+细胞体外诱导并扩增巨核祖细胞体系中，以TPO/FL/IL-6/IL-3组合的扩增效果最好，明显高于其它3组，CD41+细胞第5天、10天分别扩增了93.97±17.27倍、131.23±18.26倍。第15天CD41+细胞含量及CD41+细胞数迅速下降。CFU-MK产率（/1×103个细胞）第5天、10天分别为83.33±10.02个、120.67±13.01个，明显高于其余3组。结论：以TPO/FL/IL-6/IL-3因子组合为体外诱导扩增巨核祖细胞的最佳组合，动员外周血的巨核祖细胞体外诱导扩增以培养第10天为宜。本实验建立了动员人外周血来源的巨核祖细胞体外扩增体系。 【关键词】 动员人外周血； 巨核祖细胞; 体外扩增； 　　Ex vivo Expansion of Megakaryocytic Progenitors from Mobilized Human Peripheral Blood 　　Abstract This study was aimed to investigate the effect of some culture system compased of various cytokine combinations (TPO，SCF，FL，IL-1，IL-3，IL-6) on ex vivo expansion of megakaryocytic progenitors inducced from CD34+ cells of peripheral blood and to seek a optimal cytokine combination and culture time. Mononuclear cells were isolated from mobilized peripheral blood (MPB) by density gradient centrifugation over Ficoll. CD34+ cells were purified by using an immunomagnetic bead separation system. The selected CD34+ cells were seeded in Iscove′ s modified Kulbecco′ s medium (IMDM) supplemented with fetal calf serum (FCS) and various kinds of cytokines. After 15 days of culture，the content of CD41+ cells in culture system were determined by flow cytometry，and the number of megakaryocyte colony-forming unit (CFU-MK) was measured simultaneously. The results showed that after definited days of culture，the cytokine combination TPO/FL/IL-6/IL-3 was the most suitable for MPB to obtain high number of MK，and better than any other three groups（0.05）. The increase multiple of CD41+ cells was 93.97±17.27 on day 5 and 131.23±18.26 on day 10. On day 15，the proportion and the increase multiple of