谷氨酸对胚胎大鼠神经干细胞巢蛋白表达的影响

Effect of glutamic acid on the nestin protein expression of neural stem cells from embryonic rat

目的探讨谷氨酸对体外培养的大鼠神经干细胞巢蛋白(Nestin)的影响。方法取怀孕10 d的SD大鼠胚胎海马组织,分离神经干细胞进行体外培养,置于含碱性成纤维细胞生长因子(bFGF)、表皮细胞生长因子(EGF)、B27的无血清培养基进行培养。传代至第2代,用免疫荧光细胞化学法检测神经干细胞特异性标志物Nestin的表达;培养液中添加5-溴脱氧尿嘧啶核苷(BrdU)3 h后检测其在神经干细胞中的表达;含血清不含神经生长因子的培养液中培养7 d后经神经胶质酸性蛋白(GFAP)免疫荧光细胞化学法检测细胞分化情况。细胞传至第3代细胞后,经细胞计数后分为6组,A组为对照组,在B、C、D、E、F组培养液中分别加入浓度为200、400、600、800及1 000μmol/L的谷氨酸。观察各组神经干细胞的增殖情况,应用聚合酶链式反应(PCR)技术检测各组中Nestin表达量的不同,应用统计学软件进行分析。结果 (1)细胞培养:胎鼠海马区分离培养的神经干细胞能很快增殖形成悬浮生长的圆形细胞球。经免疫荧光鉴定,细胞球Nestin抗体标记阳性。在培养液中加入BrdU后,BrdU能结合到细胞核中;诱导分化后,荧光鉴定GFAP标记阳性。(2)逆转录-聚合酶链反应(RT-PCR):Nestin mRNA在各组之间表达亦具有显著差异(P<0.05),其表达程度由高到低顺序与谷氨酸在各组中的浓度高低相一致:F组(2.21±0.42)、E组(1.99±0.37)、D组(1.58±0.26)、C组(1.36±0.41)、B组(1.05±0.34)、A对照组(0.83±0.21)。结论谷氨酸可以促进神经干细胞Nestin的表达,且在一定范围内表达量与谷氨酸浓度存在剂量依赖性。

Objective To investigate the effects of glutamic on the nestin protein expression of cultured neural stem cells （ NSCs ） , and to provide a theoretical basis to further clarify the signaling pathways of proliferation and differentiation of NSCs and clinical nerve regeneration after injury. Methods The NSCs obtained from subgranular zone （SGZ） of mice＇ s embryon were cultured in the serum-free culture medium, which was added basic fibroblast growth factor （bFGF） and epidermal growth factor （EGF）. Then the fluorescence immunocytochemistry was adopted to detect the expression of NSCs specific factor Nestin, the proliferation of NSCs were also detected by 5-Bromo-2-deoxy Uridine （ BrdU ） method; The differentiation of NSCs were determined by the specific marker glial fibrillary acidic protein（GFAP）, which was detected after 7 days treatment by the cell culture medium containing 10% FBS. After 3 passages, the NSCs were assigned to six groups （group A, B, C, D, E, F）. Group A was control group, and group B, C, D, E, F were treated by gradient concentrations of glutamic, the proliferation of each group was detectd and polymerase chain reaction（PCR） was performed to identify the proliferation result of NSCs. The group difference was determined by statistics. Results （ 1 ） Cell culture： the NSCs, isolated from the embryonic mice＇ s cerebral cortex, were successfully cultured and could continuously proliferate and then formed the specific neurospheres. The results of immunofluorescence showed that both of the primary and the passaged neurospheres were all Nestin positive. The GFAP or BrdU positive were also detected in the differentiated neural stem cells. （2）Reverse transcription-polymerase chain reaction （RT-PCR） ：The significant difference of Nestin mRNA expression existed in various groups （ P 〈 0.05 ）, the extent of it＇ s expression showed decreasing gradually ： Group F（2.21 ± 0.42 ）, Group E （ 1.99 ± 0.37 ）, Group D （ 1.58 ± 0.26 ）, Group C （ 1.36± 0.41 ）, Group B （ 1.05 ± 0.34）, Group A （0.83± 0.21 ）. Conclutions Nestin mRNA in the glutamic group and control group were expressed, and its expression level increased with the higher concentration of glutamic.