人脐血单个核细胞分离、培养方法的比较

发布时间：2018-12-08 19:53

【摘要】： 目的:采用不同的分离、培养条件,以筛选出一种相对较好的脐血单个核细胞(mononuclear cells ,MNCs)分离、培养方法。方法:取2007-5/2008-2新疆医科大学第一附属医院产科健康产妇的脐血,比较不同分离方法所得脐血MNCs数及随后的细胞形态学变化,比较不同换液时间、不同培养基、不同浓度的胎牛血清对脐血MNCs原代培养及传代过程的影响,在原代培养第30天通过流式细胞仪检测细胞表面免疫表型。结果:3种分离方法在分离脐血MNCs数方面差异具有统计学意义(P0.01),其中以羟乙基淀粉沉淀法得到的MNCs数量最多(15.23±4.30)×106/mL,密度梯度离心法、CD133免疫磁珠组所获MNCs数分别为(3.71±1.14)×106/mL、(0.066±0.027)×106/mL。但羟乙基淀粉沉淀法所得细胞生长状态欠佳。首次第5天～7天换液、低糖DMEM、IMDM培养基有利于MNCs的生长。还不能认为30%胎牛血清与20%胎牛血清的培养基在培养脐血MNCs方面有差别。流式检测密度梯度离心法、CD133免疫磁珠组CD34阳性率分别为10.1%、0.5%。结论:羟乙基淀粉沉淀法是一种高效的脐血分离方法,但其所得细胞的生长状态欠佳,需进一步实验证实。CD133免疫磁珠法所获细胞纯度高,可根据不同要求选用相应分离法。首次第5天～7天换液、低糖DMEM、IMDM培养基是相对较好的MNCs培养条件。30%胎牛血清与20%胎牛血清的培养基在培养脐血MNCs方面尚未发现差别。
[Abstract]:Objective: to select a relatively good method for (mononuclear cells, MNCs) isolation and culture of umbilical cord blood mononuclear cells (UCB) by using different isolation and culture conditions. Methods: umbilical cord blood was collected from healthy parturients in the first affiliated Hospital of Xinjiang Medical University from May 2007 to February 2008. The number of umbilical cord blood (MNCs) obtained from different isolation methods and the subsequent morphological changes were compared, and the time of fluid exchange and different media were compared. The effects of different concentrations of fetal bovine serum on the primary culture and passage of umbilical cord blood MNCs were investigated by flow cytometry on the 30th day of primary culture. Results: there were significant differences among the three methods in the number of MNCs isolated from umbilical blood (P0.01), in which the MNCs obtained by hydroxyethyl starch precipitation method was the largest (15.23 卤4.30) 脳 106 mL, and the density gradient centrifugation method was used. The MNCs numbers in CD133 immunomagnetic beads group were (3.71 卤1.14) 脳 10 ~ (6) / mL and (0.066 卤0.027) 脳 10 ~ (-6) / mL, respectively. However, the growth state of the cells obtained by hydroxyethyl starch precipitation was not good. For the first 5 ~ 7 days, the low sugar DMEM,IMDM medium was beneficial to the growth of MNCs. The culture medium of 30% fetal bovine serum and 20% fetal bovine serum could not be considered to be different in the culture of MNCs in umbilical cord blood. The positive rates of CD34 in CD133 immunomagnetic beads group were 10. 1% and 0. 5% respectively. Conclusion: hydroxyethyl starch precipitation method is an effective method for the separation of umbilical cord blood, but the growth state of the cells obtained is poor, which needs to be confirmed by further experiments. The CD133 immunomagnetic beads method has high purity and can be separated according to different requirements. For the first 5 ~ 7 days, low glucose DMEM,IMDM medium was a relatively good medium for MNCs culture, and there was no difference between 30% fetal bovine serum medium and 20% fetal bovine serum medium in the culture of umbilical cord blood MNCs.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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