胎鼠不同脑组织中神经干细胞和神经元所占比例及差异

发布时间：2019-05-27 20:02

【摘要】：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(P0.01)和大脑皮质(P0.05);而前脑组织中Tuj1~+细胞比例明显低于全脑(P0.05)和大脑皮质(P0.001)。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(P0.05),略高于大脑皮质(P0.05),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(P0.05)和大脑皮质组织(P0.05),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。
[Abstract]:Background: at present, the in vitro culture technology of mouse neural stem cells has been skillfully mastered by many scientific research platforms, but the brain tissue used by each scientific research platform in the culture of brain-derived neural stem cells is very different. The brain tissue of 14-day-old (E14) mice is widely used to culture fetal rat neural stem cells, but there are few studies on the detailed proportion of neural stem cells in different brain tissues of fetal rats at this time and whether there is any difference between them. Aim: to compare the proportion and difference of neural stem cells and neurons in the whole brain, cerebral cortex and forebrain of E14 mice, and to provide direct quantitative data and preliminary research basis for the optimal isolation of high purity neural stem cells in the later stage. Methods: the whole brain, cerebral cortex and forebrain of E14 mice were isolated and digested into single cells with trypsin. The cells were cultured for 3.0 h and 4.0 h, and then the total cells were labeled with DAPI. Neural stem cell specific antigen Nestin labeled neural stem cell and neuron specific antigen Tuj1 labeled neuron. The proportion of Nestin~ / DAPI and Tuj1~ / DAPI was statistically analyzed. In addition, the expression of Nestin and Tuj1 mR NA in the whole brain, cerebral cortex and forebrain of E14 mice was detected by real-time fluorescence quantitative PCR. Results and conclusion: (1) the results of cellular immunofluorescence showed that there were a large number of Nestin~ and Tuj1~ cells in the whole brain, cerebral cortex and forebrain of E14 mice. The proportion of Nestin~ cells in forebrain tissue of mice was the highest, which was significantly higher than that in whole brain (P 0.01) and cerebral cortex (P 0.05), while the proportion of Tuj1~ cells in forebrain tissue was significantly lower than that in whole brain (P 0.05) and cerebral cortex (P0.001). The results showed that compared with the whole brain and cerebral cortex, the proportion of neural stem cells in forebrain tissue was the highest and the proportion of neurons was the lowest. (2) the results of) Real time PCR showed that the expression level of Nestin mR NA in forebrain tissue of E14 mice was the highest, which was significantly higher than that of whole brain tissue (P 0.05) and slightly higher than that of cerebral cortex (P 0.05), which indicated that there were more neural stem cells in the forebrain tissue of E14 mice. The expression level of Tuj1 mR NA was significantly lower than that of whole brain tissue (P 0.05) and cerebral cortex tissue (P 0.05), indicating that there were fewer neurons in the anterior brain tissue. (3) compared with the whole brain and cerebral cortex, the proportion of neural stem cells obtained by isolating and digesting the anterior brain tissue of E14 mice was the highest, which was an important prerequisite for the later culture of high quality neural stem cells.
【作者单位】：昆明医科大学分子临床医学研究院;深圳市宝安区人民医院;澳大利亚莫纳什大学免疫与干细胞研究中心;
【基金】：云南省高端人才引进计划(20080A004) 云南省干细胞和再生医学重点实验室(2015DG027)~~
【分类号】：R318

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