| 董玲凤 余瑾 赵艳 李自立 窦丹丹 胡亮.两种不同密度分离液分离的脐血CD34+、CD34-细胞造血活性比较[J].,2016,16(1):11-15 |
| 两种不同密度分离液分离的脐血CD34+、CD34-细胞造血活性比较 |
| Comparison of Hematopoietic Activities of Umbilical Cord Blood CD34Positive and Negative Cells fromMNCs Separated on Two DifferentFicoll-urografin Gradients |
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| DOI： |
| 中文关键词: 脐血 造血干、祖细胞 单个核细胞 Ficoll-泛影葡胺分离液 密度梯度离心 |
| 英文关键词: Umbilical cord blood Hematopoietic stem/ progenitor cells Mononuclear cells Ficoll-urografin separation media Density
gradient centrifugation |
| 基金项目:国家自然科学基金项目（81372627）；湖南省自然科学基金项目（13JJ3038） |
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| 中文摘要: |
| 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/mL、(1.077± 0.001) g/mL 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-depletedMNCs 在甲基纤维素中形成CFU-GM集落数。结果：① (1.068±
0.001) g/mL、(1.077± 0.001) g/mL 两种分离液分离的单个核细胞平均密度分别为（1.52± 1.0）× 106个/mL、(2.84± 0.98）× 106个
/mL，（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/mL分离的单个核细胞经免疫磁珠纯化后1.5× 103个CD34+细胞形成CFU-GM的集落(140.5± 14.5) 显著多于
(1.077± 0.001) g/mL 的集落数(118.3± 13.8)（P< 0.05）；(1.068± 0.001) g/mL 分离的5× 104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/mL 的集落数(101.3± 9.4 )，（P< 0.05）。结论：与1.077 g/mL Ficoll 相比，1.068
g/mL 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。 |
| 英文摘要: |
| Objective:To find a better concentration of ficoll-urografin for isolating hematopoietic stem cells (HSCs) and
hematopoietic progenitor cell （HPCs）from umbilical cord blood in medical research and clinical application.Methods:Six umbilical
cord blood samples were collected from the hospital. The separation media with two kinds of concentrations, i.e. (1.068± 0.001) and
(1.077± 0.001) g/mL, were used to isolate the mononuclear cells in the same sample by using density gradient centrifugation method.
The harvest rate and the survival rate of mononuclear cells were counted and analyzed. High purity CD34+cells and CD34-depleted MNCs
were obtained by Magnetic Activated Cell Sorting (MACS). Finally, CD34+cells and CD34-depletedMNCs separated by two density separation
media was cultured in methylcellulose and the colony -forming unit -granulocyte macrophage (CFU-GM) was counted to evaluate
their hematopoietic potential.Results:①With the two different concentrations of separation media (1.068± 0.001) and (1.077± 0.001)
g/mL,（1.52± 1.0）× 106/mL and (2.84± 0.98)× 106/mL MNCs could be obtained respectively, the number of MNCs with the two separation
media has no significant difference (P>0.05); the percentage of CD34+ cells in mononuclear cells were（1.26± 0.47）%,（1.07±
0.15）% respectively, (no significant difference, P>0.05). ②The clone number of CFU-GMfrom1.5× 103 CD34+ cells separated with the
concentration of (1.068± 0.001) g/mL was significantly higher than that with (1.077± 0.001) g/mL separation media (140.5± 14.5 vs.
118.3± 13.8, P< 0.05). The clone number of CFU-GM from 5× 104 CD34-depleted MNCs with the concentration of (1.068± 0.001) g/mL
was also significantly higher than that with concentration of (1.077± 0.001) g/mL (132.0± 5.1 vs. 101.3± 9.4, P<0.05).Conclusion:Compared to the group with (1.077± 0.001) g/mL Ficoll-Hypaque, the separation medium with the concentration (1.068± 0.001) g/mL could relatively enrich mononuclear cells with higher hematopoietic potential, which means that separation medium with the concentration
(1.068± 0.001) g/mL may be better to isolate hematopoietic stem/progenitor cells fromumbilical cord blood. |
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