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目的:比较两种不同密度的ficoll-泛影葡胺分离造血干细胞群的效果,找出更适合分离脐血造血干、祖细胞的分离密度,为脐血造血干细胞的医学研究及临床应用提供一定依据。方法:采集脐血6份,根据密度梯度离心法,用质量分数为(1.068±0.001)g/m L、(1.077±0.001)g/m L ficoll-泛影葡胺分离液等量分离同一份脐血,分别计数这两种分离液的单个核细胞获得率及细胞存活率,得到的单个核细胞再分别经免疫磁珠分选法获得高纯度的CD34~+细胞及除去了CD34~+的单个核细胞(CD34~(-depleted) MNCs),统计分析这两种不同密度分离液获得的CD34~+细胞、CD34~(-depleted)MNCs在甲基纤维素中形成CFU-GM集落数。结果:1(1.068±0.001)g/m L、(1.077±0.001)g/m L两种分离液分离的单个核细胞平均密度分别为(1.52±1.0)×10~6个/m L、(2.84±0.98)×10~6个/m L,(P>0.05);经免疫磁珠纯化后的CD34~+细胞占单个核细胞(MNCs)的比率分别是(1.26±0.47)%、(1.07±0.15)%,(P>0.05);2(1.068±0.001)g/m L分离的单个核细胞经免疫磁珠纯化后1.5×10~3个CD34~+细胞形成CFU-GM的集落(140.5±14.5)显著多于(1.077±0.001)g/m L的集落数(118.3±13.8)(P<0.05);(1.068±0.001)g/m L分离的5×10~4个CD34~(-depleted) MNCs形成的CFU-GM集落数(132.0±5.1)也显著多于(1.077±0.001)g/m L的集落数(101.3±9.4),(P<0.05)。结论:与1.077 g/m L Ficoll相比,1.068g/m L Ficoll-泛影葡胺分离液分离的单个核细胞中的CD34~+、CD34~-细胞造血活性更强,更适合用来分离脐血中造血干细胞群。
OBJECTIVE: To compare the effect of two different densities of ficoll-diatrizoate on the isolation of hematopoietic stem cell population and to find out the more suitable isolation of hematopoietic stem and progenitor cells from cord blood, so as to provide certain medical research and clinical application of cord blood hematopoietic stem cells in accordance with. Methods: 6 samples of umbilical cord blood were collected, and the same fraction was separated by density gradient centrifugation with the same amount of (1.068 ± 0.001) g / m L, (1.077 ± 0.001) g / m L ficoll-diatrizoate Umbilical cord blood were counted, respectively, the two separation fluid obtained mononuclear cells and cell viability, the resulting mononuclear cells and then by magnetic separation method to obtain high purity CD34 ~ + cells and remove the CD34 ~ + Mononuclear cells (CD34 ~ (MNCs)) were counted and the CD34 + cells and CD34 + MNCs obtained from these two different densities were statistically analyzed for the number of CFU-GM colonies in methylcellulose. Results: The average density of mononuclear cells isolated from 1 (1.068 ± 0.001) g / m L and (1.077 ± 0.001) g / m L separated from each other was (1.52 ± 1.0) × 10 6 / m L 2.84 ± 0.98) × 10-6 / m L, (P> 0.05). The percentage of CD34 + cells and mononuclear cells (MNCs) purified by immunomagnetic beads were (1.26 ± 0.47)% and ± 0.15%), (P> 0.05). The number of CFU-GM-producing colonies (140.5%) in 1.5 × 10 ~ 3 CD34 ~ + cells purified by immunomagnetic beads after 2 (1.068 ± 0.001) g / ± 14.5) was significantly higher than that of (1.077 ± 0.001) g / m L (118.3 ± 13.8) (P <0.05) depleted MNCs formed more CFU-GM colonies (132.0 ± 5.1) than those (1.077 ± 0.001) g / m L colonies (101.3 ± 9.4) (P <0.05). CONCLUSION: Compared with 1.077 g / ml Ficoll, CD34 +, CD34 ~ - cells in mononuclear cells isolated from 1.068 g / ml Ficoll-diatrizoate were stronger and more suitable for separation Cord blood hematopoietic stem cell population.