摘要
目的优化实验设计将人神经干细胞(h NSCs)高比例诱导分化为多巴胺能神经元。方法从a FGF、dbcAMP、普拉克索(PRX)、TPA和forskolin共计5种诱导因子中,使用单因素方法筛选出对h NSCs诱导为多巴胺能神经元有显著作用的诱导因子,显著性检验使用student's t检验。之后使用析因设计方法对选出的显著因子的诱导作用进行定量评估,找出每个因子对h NSCs诱导为多巴胺能神经元的诱导规律,统计学显著性使用方差分析。最后使用最快上升法找到每个显著因子的最佳浓度。结果根据初步的单因素筛选实验,aFGF、dbcAMP、TPA被认为是对h NSCs诱导为多巴胺能神经元起诱导作用的因子。使用析因设计对aFGF、dbcAMP和TPA的因子效应进行建模,得出在初始的浓度下,aFGF和dbcAMP为阳性诱导因子,TPA为阴性诱导因子,并得出三者的浓度变化比例为0.29:0.90:-0.39。选取三者的步长分别为11 ng/ml、35μmol/L和-15 nmol/L,使用最快上升法优化出三者最佳的浓度分别为166 ng/ml、310μmol/L和10 nmol/L。最终多巴胺能神经元诱导比例可以达到(6.56±0.07)﹪,相比较对照提高了23.8﹪。结论析因设计法可以有效的优化h NSCs诱导分化为多巴胺能神经元。
Objective To induce the differentiation of human neural stem cells （hNSCs） into dopaminergic neurons. Methods From a total of 5 kinds of inducing factors including aFGF, dbcAMP, pramipexole （PRX）, TPA （phorbol-12-myristae-13-acetate）, forskolin, single factor experiments were used to screen significant factors which play an important role in inducing hNSCs into dopaminergic neurons, and significance tests were tested by student＇s t. Factorial design was used to evaluate the inducing effect of the selected significant factors and to find out how these factors affect the induced dopaminergic neuron positive ratio. Statistical significance was tested by analysis of variance. Finally, the optimal concentration of each significant factor was determined by using the steepest-ascent method. Results According to single factor experiments, aFGF, dbcAMP and TPA were considered to be important factors in inducing differentiation of dopaminergic neurons form hNSCs. Inducing effect of aFGF, dbcAMP and TPA was modeled by factorial design. These results showed that aFGF and dbcAMP were positive factors and TPA was negative factor at the initial concentrations. The ratio of concentration changes in aFGF, dbcAMP and TPA should be 0.29 ： 0.90 ： -0.39. The stepwise of aFGF, dbcAMP was selected as 11 ng/ml, 35 ~tmol/L and -15 nmol/L, and TPA used in the steepest-ascent method respectively. Then, the optimal concentration of aFGF, dbcAMP and TPA was achieved at 166 ng/ml, 310 ktmol/L and 10 nmol/L. The optimal dopaminergic neuron positive ratio was （6.56±0.07） %, which increased by 23.8 % comparing to control group. Conelusion Factorial design can effectively optimize the differentiation of hNSCs into dopaminergic neurons.
出处
《中华细胞与干细胞杂志（电子版）》
2017年第4期195-201,共7页
Chinese Journal of Cell and Stem Cell（Electronic Edition）
基金
山东省2016年重点研发计划(创新型产业集群)项目(2016ZDJQ0104)
泰山学者建设工程专项经费(tshw201502002)
