胎鼠不同脑组织中神经干细胞和神经元所占比例及差异

Proportion and difference of neural stem cells and neurons from different embryonic mouse brain tissues

背景:目前,小鼠神经干细胞的体外培养技术已被众多科研平台熟练掌握,但是各科研平台在培养脑源性神经干细胞时所使用的脑组织有很大差异。胎龄14 d(E14)小鼠脑组织被广泛用于培养胎鼠神经干细胞,而对于此时胎鼠不同脑组织中所含神经干细胞的详细比例及其间有无差异,目前研究较少。目的:比较E14小鼠全脑、大脑皮质和前脑组织中神经干细胞和神经元所占比例及差异,为后期优化分离高纯度神经干细胞提供直接量化数据和前期研究基础。方法:分离E14小鼠全脑、大脑皮质和前脑组织,加入胰酶消化成单细胞,贴壁培养3.0-4.0 h,然后通过细胞免疫荧光实验,用DAPI标记总细胞,神经干细胞特异性抗原Nestin标记神经干细胞,神经元特异性抗原Tuj1标记神经元,统计分析Nestin^+/DAPI和Tuj1^+/DAPI所占比例。另外,通过实时荧光定量PCR技术检测E14小鼠全脑、大脑皮质和前脑组织中Nestin和Tuj1 mR NA的表达水平。结果与结论:(1)细胞免疫荧光结果显示,E14小鼠全脑、大脑皮质和前脑组织中均有大量Nestin^+和Tuj1^+细胞存在;小鼠前脑组织中Nestin^+细胞所占比例最高,明显高于全脑(P<0.01)和大脑皮质(P<0.05);而前脑组织中Tuj1^+细胞比例明显低于全脑(P<0.05)和大脑皮质(P<0.001)。该结果表明,与全脑和大脑皮质相比,前脑组织中神经干细胞所占比例最高,神经元所占比例最低;(2)Real time PCR结果显示,E14小鼠前脑组织中Nestin mR NA表达水平最高,明显高于全脑组织(P<0.05),略高于大脑皮质(P>0.05),说明前脑组织中有较多神经干细胞存在;而Tuj1 mR NA的表达水平明显低于全脑组织(P<0.05)和大脑皮质组织(P<0.05),说明前脑组织中有较少神经元存在;(3)综合以上结果,与全脑和大脑皮质相比,分离、消化E14小鼠前脑组织所获得的神经干细胞比例最高,是后期培养优质神经干细胞的重要前提。

BACKGROUND：At present, mouse embryonic neural stem cells （NSCs） culture has been skillfully operated by many labs, but there are differences existing about which part are dissociated to get NSCs. Embryonic 14 days （E14） mouse brain tissues are widely used for culturing NSCs, but there are less studies about the detailed percentage and difference of NSCs separated from different brain tissues. OBJECTIVE： To test the proportion and difference of NSCs and neurons percentage from E14 mouse whole brain, cortex and forebrain, providing quantized data for optimizing the isolation of high-purity NSCs. METHODS：E14 C57BL/6 mouse whole brain, cortex and forebrain tissues were separated and dissociated into single cells that were adherently cultured for 3.0-4.0 hours and labeled by DAPI. Then the cells were immunostained with NSCs specific marker, Nestin, and neuron specific marker, Tuj1, to identify NSCs and neurons percentage by calculating Nestin＋/DAPI and Tuj1＋/DAPI. In addition, real-time PCR assay was used to test Nestin and Tuj1 mRNA expression in the E14 mouse whole brain, cortex and forebrain. RESULTS AND CONCLUSION： （1） Immunocytochemical results showed that there were a large amount of Nestin＋ and Tuj1＋ cells in the whole brain, cortex and forebrain of E14 mice. NSCs percentage in the forebrain was obviously higher than that in the whole brain （P ＆lt; 0.01） and cortex （P ＆lt; 0.05）, while the percentage of neurons in the forebrain was significantly lower than that in the whole brain （P ＆lt; 0.05） and in the cortex （P ＆lt; 0.001）. （2） Real-time PCR results showed that the Nestin mRNA expression in the forebrain was significantly higher than that in the whole brain （P ＆lt; 0.05） and slightly higher than that in the cortex （P ＆gt; 0.05）; the Tuj1 mRNA expression in the forebrain was significantly lower than that in the whole brain （P ＆lt; 0.05） and in the cortex （P ＆lt; 0.05）. These findings indicated that the forebrain had the most NSCs and the least neurons compared with the whole brain and the cortex. In summary, E14 mouse forebrain has the highest percentage of NSCs compared with the whole brain and cortex, which is a better source to obtain NSCs for the following cell culture experiments.