摘要
目的观察非必需氨基酸（NEAA）和β-巯基乙醇对神经干细胞（NSCs）增殖的影响。方法取孕龄为14.5 d的雌鼠,处死后提取NSCs并培养,取5~7代的NSCs用于实验,将NSCs细胞使用传代培养基培养,并分为四组,对照组：加入磷酸盐缓冲液（PBS）12μL;NEAA组：加入NEAA 10μL,PBS 2μL;β-巯基乙醇组：加入β-巯基乙醇2μL,PBS 10μL;NEAA＋β-巯基乙醇组：加入NEAA 10μL,β-巯基乙醇2μL。通过细胞计数法计算NSCs的增殖速度,噻唑蓝（MTT）法测定NSCs增殖率,RT-PCR法测定NSCs中凋亡相关基因含半胱氨酸的天冬氨酸蛋白水解酶-3（Caspase-3）的表达,免疫组化法对NSCs进行鉴定并测定NSCs向神经元和星形胶质细胞的分化潜能。结果与对照组比较,NEAA组NSCs增殖速度、增殖率升高,Caspase-3 mRNA降低（P〈0.05或〈0.01）;β-巯基乙醇组及NEAA＋β-巯基乙醇组NSCs增殖速度、增殖率升高,Caspase-3 mRNA、TUJ-1阳性细胞百分比、GFAP阳性细胞百分比降低,Nestin升高（P〈0.05或〈0.01）。与NEAA组比较,NEAA＋β-巯基乙醇组各项指标差异均有统计学意义（P〈0.05或〈0.01）;与β-巯基乙醇组比较,NEAA＋β-巯基乙醇组NSCs增殖速度、增殖率升高,Caspase-3mRNA降低（P〈0.05或〈0.01）。结论 NEAA和β-巯基乙醇均能促进NSCs增殖,联合使用起协同作用。
Objective To investigate the effects of combined application of non-essential amino acids（ NEAA） and β-mercaptoethanol on proliferation of neural stem cells（ NSCs）. Methods The female rats in the gestational age of 14. 5 d were selected and executed to extract NSCs and cultivate. We took 5-7 generations of NSCs to do the experiments. The NSCs cultured were divided into four groups： the control group which was added with 12 μL phosphate buffer solution（ PBS）,the NEAA group which was added with 10 L NEAA and 2 L PBS,the β-mercaptoethanol group which was added with 2 μL β-mercaptoethanol and10 L PBS,and the NEAA ＋ β-mercaptoethanol group which was added with 10 L NEAA and 2 μL β-mercaptoethanol. The proliferation of NSCs was calculated by cytometry,the proliferation rate of NSCs was detected by MTT,the caspase-3 expression of NSCs was assayed by RT-PCR,and NSCs were identified and examined for the differentiation potential of NSCs into neurons and astrocytes by immunohistochemistry. Results Compared with the control group,the proliferation speed and rate of NSCs in the NEAA group were increased,but the caspase-3 mRNA expression of NSCs was decreased（ P〈0. 05 or P〈0. 01）; the proliferation speed and rate of NSCs were increased,but the caspase-3 mRNA expression of NSCs,the percentage of positive ATUJ-1cells,the percentage of positive GFAP cells were decreased and Nestin was increased in the β-mercaptoethanol group and NEAA＋ β-mercaptoethanol group（ P〈0. 05 or P〈0. 01）. Significant difference was found in each index between the NEAA group and NEAA ＋ β-mercaptoethanol group（ P〈0. 05 or P〈0. 01）. Compared with the β-mercaptoethanol group,the proliferation speed and rate of NSCs were increased,but the caspase-3 mRNA expression of NSCs was decreased in the NEAA ＋ β-mercaptoethanol group（ P〈0. 05 or P〈0. 01） Conclusion Both NEAA and β-mercaptoethanol can promote the proliferation of neural stem cells and they have synergistic effect during the combined application.
出处
《山东医药》
CAS
北大核心
2016年第1期21-23,共3页
Shandong Medical Journal
基金
国家自然科学基金资助项目(31460314)
关键词
神经干细胞
非必需氨基酸
β-巯基乙醇
增殖
分化
neural stem cells
non-essential amino acids
β-mercaptoethanol
proliferation
differentiation
