脐血单核细胞的优化培养

Experimental study of optimized culture of umbilical cord blood.mononuclear cells

目的优化脐血单核细胞（UCB-MNCs）的培养方法。方法采用正交实验方法筛选影响UCB．MNCs培养的各因素，优化培养条件。设立优化组、对照组（UCB-MNCs以1．0×10^6／L接种于含20μg/L碱性成纤维细胞生长因子（bFGF）、体积分数为0．10胎牛血清、低糖DMEM培养基）培养。其后检测各组培养细胞增殖及形态学变化；流式细胞仪对各组行免疫表型分析。结果筛选后优化培养条件为：低糖DMEM培养基中含20mg／LbFGF、50μg／L干细胞因子（SCF）、10峙／L重组人粒细胞生长因子（G—CSF）、培养瓶用层粘连蛋白（LN）包被。原代培养的细胞密度优化组细胞（108．15±6．90）增殖优于对照组（51．48±12．27），差异有统计学意义（P〈0．01）。P3代UCB—MNCs中MSCs含量优化组（94．87％）优于对照组（75．35％），差异有统计学意义（P〈0．05）。结论采用正交实验的方法可以筛选出脐血单核细胞的优化培养条件。

Objective To optimize the culture method of umbilical cord blood-mononuclear cells （UCB-MNCs）. Methods First, crosscut empirical method was employed to sieve culture factors of UCB-MNCs, optimize the culture condition. Set up optimize group and control group [ UCB-MNCs was plated in 1.0 ×10^6/L Lo CHO DMEM supplemented with 20 I.Lg/L basic fibroblast growth factor （bFGF）, 10% FBS）]. Then generation and morphology was detected. Immunophenotype was analysesed by FCM. Results The sieved optimized cultivate condition： culture flask was coating by laminin （LN）, UCB-MNCs was plated in it （ 1.0 ×10^6/L） containing Lo CHO DMEM supplemented with 10 μg/L granulocyte colony stimulating factor （G-CSF）, 50 p.g/L stem cell factor （SCF）, 20 μg/L bFGF, 20 ml/L B27. Optimize group （ 108. 15 ±6. 90） Compared to the control group （51.48 ± 12. 27） , cell proliferation was more significant （P 〈0. 01 ）, P3 UCB-MNCs content more MSCs in optimized group. Conclusion Optimized cultivate condition of UCB-MNCs could be sieved by orthogonal experiment.