无糖对体外培养的神经前体细胞损伤的影响

Effect of glucose-free treatment on the neural precursor cells in vitro

目的 :研究无糖处理对体外分离培养神经前体细胞 (neuralprecursorcells,NPCs)的存活、分裂及分化能力的影响。方法 :培养的第 4～ 6代新生大鼠海马区NPCs给予无糖培养基处理 6h后 ,分别于处理后第 0、1和 7天测定细胞MTT代谢率、台盼蓝染色细胞率、5′ 溴 脱氧尿嘧啶 (5′ bromo deoxyuridine,BrdU)参入率及分化细胞内胶质细胞纤维酸性蛋白、神经元特异性烯醇酶和半乳糖脑苷蛋白表达 ,判定NPCs存活、分裂和分化能力的改变。结果 :(1 )无糖处理后的不同时间点台盼蓝染色阳性细胞率在NPCs无糖组较NPCs对照组增加。无糖处理后即刻和 1d ,NPCs无糖组较神经元无糖组减少 ,7d时两者相近。 (2 )无糖损伤后 0d和 1d ,NPCs无糖组的MTT代谢率较NPCs对照组明显降低 (P <0 .0 5 ) ,7d时差异无显著性 (P >0 .0 5 )。在不同时间点 ,NPCs无糖组的MTT代谢率较神经元无糖组均明显增高 (P <0 .0 5 )。 (3)无糖处理后 ,NPCs无糖组的BrdU参入率与对照组相比 ,差异无显著性。 (4 )无糖对NPCs分化成为神经元、星形胶质细胞和少突胶质细胞的比例无显著性影响 (P >0 .0 5 )。结论 :无糖可以损伤NPCs 。

Objective: To investigate the survival, division, and differentiation of neural precursor cells (NPCs) following glucose free treatment in vitro . Methods: Cultured neonatal rat hippocampal NPCs were exposed to glucose free media for 6 hours, and then were returned to regular media containing normal level glucose. At different time points after glucose free treatment, trypan blue staining was used to determine cell viability, MTT assay to measure cell metabolic rate, 5′ bromo deoxyuridine (BrdU) incorporation to determine the NPCs division, and immunocellulochemistry and western blot analysis to study the NPCs differentiation. Results: (1)At different time points (0, 1, 7 d) after glucose free treatment, NPCs viability decreased compared with NPCs control. On 0 and 1 d after glucose free treatment, NPCs viability was higher than neurons viability. (2) On 0 and 1 d after glucose free treatment, NPCs metabolic rates decreased significantly compared with NPCs control. On 0, 1, and 7 d after glucose free treatment, NPCs metabolic rates were higher than that in neurons. (3) After glucose free treatment, BrdU incorporation of NPCs was similar to NPCs control ( P >0.05). (4) After glucose free treatment, NPCs differentiation didn't change significantly ( P >0.05). Conclusion: These findings demonstrated that glucose free treatment impaired NPC’s viability, but didn’t affect the division and differentiation of NPCs.