葡萄糖浓度对原代培养神经干细胞增殖、分化的影响

Effect of glucose concentration on proliferation and differentiation of neural stem cells in primary culture

目的探讨不同浓度葡萄糖对神经干细胞增殖、分化的影响。方法胚胎小鼠脑细胞原代培养并鉴定。生理浓度和高浓度葡萄糖培养及诱导分化后,四唑盐比色(MTT)法及免疫荧光法观察两组不同葡萄糖浓度对神经干细胞增殖、分化的影响。结果原代培养的神经球表达巢蛋白(nestin),神经球分化的细胞表达神经丝200(NF200)和胶质纤维酸性蛋白(GFAP)。与生理浓度葡萄糖相比,高浓度葡萄糖降低了神经干细胞的增殖活力;分化3 d后,高糖组的NF200阳性细胞和GFAP阳性细胞比例高于生理浓度组(P<0.01),而nestin阳性细胞比例则反之(P<0.01);分化10 d后,神经干细胞完全分化,免疫荧光检测发现生理浓度葡萄糖提高了NF200阳性细胞比例。结论高糖损害了神经干细胞的增殖活性,加速了神经干细胞的早期分化,降低了神经元的分化率。

Objective To explore the effect of glucose at different concentrations on the proliferation and differentiation of neural stem cells （NSCs）. Methods The cells from the embryonic mouse brains were primarily cultured and identified. NSCs in the physiological concentration glucose-containing medium （1.0 g/L） and the high-glucose medium （4.5 g/L） were cultured and induced to differentiate, respectively. We observed the effects of PG and HG on the proliferation and differentiation of NSCs by MTT assay and immunofluorescence method. Results lmmunofluorescence staining showed nestin-, NF200- and glial fibrillary acidic protein （GFAP）-positive cells in neurospheres. Compared with PG, HG impaired the proliferation activity of NSCs in vitro; after 3-day differentiation, the percentages of NF200- and GFAP-positive cells were higher in the HG medium than those in the PG medium （NF200-positive cells, 17.14%±1.8% vs 13.58%±1.07%, P〈0.01; GFAP-positive cells, 59.50%±2.08% vs 47.10%±1.85%, P〈0.01）, but the percentage of nestin-positive cells was higher in the PG medium than that in the HG medium （16.05%±0.94% vs 33.47%±2.09%, P〈 0.01）; after 10-day differentiation, PG increased the proportion of NF200-positive cells. Conclusion HG impairs the proliferation activity, accelerates the early differentiation, and decreases the differentiation rate in NSCs in vitro.