利用析因设计法优化神经干细胞分化为多巴胺能神经元的诱导方案

doi:10.3877/cma.j.issn.2095-1221.2017.04.002

目的

优化实验设计将人神经干细胞（hNSCs）高比例诱导分化为多巴胺能神经元。

方法

从aFGF、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hNSCs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hNSCs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hNSCs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、35 μmol/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、310 μmol/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hNSCs诱导分化为多巴胺能神经元。

关键词: 神经干细胞, 多巴胺, 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 and TPA used in the steepest-ascent method was selected as 11 ng/ml, 35 μmol/L and -15 nmol/L, respectively. Then, the optimal concentration of aFGF, dbcAMP and TPA was achieved at 166 ng/ml, 310 μmol/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.

Conclusion

Factorial design can effectively optimize the differentiation of hNSCs into dopaminergic neurons.

Key words: Neural stem cells, Dopamine, Neurons

表1 析因设计因子水平表

因素	编码	水平
因素	编码	-1	+1
aFGF（ng/ml）	A	50	150
dbcAMP（μmol/L）	B	50	150
TPA（nmol/L）	C	50	150

表1 析因设计因子水平表

因素	编码	水平
因素	编码	-1	+1
aFGF（ng/ml）	A	50	150
dbcAMP（μmol/L）	B	50	150
TPA（nmol/L）	C	50	150

表2 析因设计及结果

标准序	运行序	aFGF（ng/ml）	dbcAMP（μmol/L）	TPA（nmol/L）	TH阳性率（﹪）
1	4	50	50	50	1.38
2	5	50	50	50	1.04
3	11	150	50	50	1.64
4	9	150	50	50	1.52
5	2	50	150	50	1.43
6	16	50	150	50	2.86
7	7	150	150	50	4.10
8	12	150	150	50	6.00
9	10	50	50	150	1.41
10	14	50	50	150	1.33
11	15	150	50	150	0.58
12	8	150	50	150	0.79
13	3	50	150	150	3.15
14	1	50	150	150	1.95
15	13	150	150	150	2.44
16	6	150	150	150	2.11

表2 析因设计及结果

标准序	运行序	aFGF（ng/ml）	dbcAMP（μmol/L）	TPA（nmol/L）	TH阳性率（﹪）
1	4	50	50	50	1.38
2	5	50	50	50	1.04
3	11	150	50	50	1.64
4	9	150	50	50	1.52
5	2	50	150	50	1.43
6	16	50	150	50	2.86
7	7	150	150	50	4.10
8	12	150	150	50	6.00
9	10	50	50	150	1.41
10	14	50	50	150	1.33
11	15	150	50	150	0.58
12	8	150	50	150	0.79
13	3	50	150	150	3.15
14	1	50	150	150	1.95
15	13	150	150	150	2.44
16	6	150	150	150	2.11

图1 光学显微镜下观察诱导因子诱导出来的β-tubulin Ⅲ和TH阳性细胞（免疫荧光染色，×400）

图2 显著因子筛选

表3 最快上升法结果

?	aFGF（ng/ml）	dbcAMP（μmol/L）	TPA（nmol/L）	TH阳性率（﹪）
第1步	100	100	100	1.48±0.10
第2步	111	135	85	1.19±0.96
第3步	122	170	70	1.32±1.86
第4步	133	205	55	2.85±1.64
第5步	144	240	40	3.46±2.48
第6步	155	275	25	5.62±0.57
第7步	166	310	10	6.56±0.07

表3 最快上升法结果

?	aFGF（ng/ml）	dbcAMP（μmol/L）	TPA（nmol/L）	TH阳性率（﹪）
第1步	100	100	100	1.48±0.10
第2步	111	135	85	1.19±0.96
第3步	122	170	70	1.32±1.86
第4步	133	205	55	2.85±1.64
第5步	144	240	40	3.46±2.48
第6步	155	275	25	5.62±0.57
第7步	166	310	10	6.56±0.07

图3 光学显微镜下观察最快上升法最佳诱导方案的β-tubulin Ⅲ和TH阳性细胞（免疫荧光染色，×400）

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Optimization of induced differentiation of human neural stem cells into dopaminergic neurons by factorial design

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Optimization of induced differentiation of human neural stem cells into dopaminergic neurons by factorial design