胎鼠原代脊髓神经元分离培养方法的改良

Improvement of primary culture method of rat spinal cord neurons

中文关键词:  脊髓神经元  原代培养  酶消化法  Accutase酶法

英文关键词:Spinal cord neuron  Primary culture  Enzyme digestion  Accutase enzyme method

中文摘要:

  【摘要】 目的：介绍一种改良原代胎鼠脊髓神经元的分离培养方法。方法：取育龄期雄、雌SD大鼠各36只，合笼配种怀孕。取受孕10~12d、13~15d及16~18d的SD大鼠各6只，解剖分离胚胎脊髓，按常规胰酶消化法制备单细胞悬液，应用细胞计数板及台盼蓝染色，在显微镜下数出蓝色细胞数和细胞总数，计算细胞存活率，明确最佳取材胎龄；接着以上述最佳取材胎龄为取材时间点，每组6只孕鼠，按无酶法、胰酶法和Accutase酶法消化三种不同的方法制备单细胞悬液，显微镜下数出蓝色细胞数和细胞总数，计算细胞存活率。接种、纯化细胞并培养1、3、7d后，用倒置相差显微镜观察脊髓神经元生长状态，利用神经元特异性标志物β tubulin Ⅲ与细胞核双标记法鉴定培养脊髓神经元的纯度。结果：孕13~15d组细胞总量和存活细胞量高于孕10~12d组，细胞存活率高于孕16~18d组（P<0.05）。Accutase酶组获取的细胞总量、存活细胞量以及细胞存活率均明显高于常规无酶组及胰酶组（P<0.05）。在倒置相差显微镜下观察发现，脊髓神经元培养7d后，胞体周围逐渐出现光圈，神经突起逐渐伸长，最终形成密集的神经突起网络；与无酶组及胰酶组相比，Accutase酶法分离的脊髓神经元分布更加均匀并且神经突起形成的网更密集。经β tubulin Ⅲ和hoechst33342荧光染色鉴定，Accutase酶组、无酶组及胰酶组三组分离提取培养的脊髓神经元纯度分别为（90.29±2.55）%、（83.51±6.20）%和（88.10±4.07）%，组间无统计学差异（P>0.05）。结论：13~15d胎龄是原代培养脊髓神经元的最佳取材时间；Accutase酶法相较于常规无酶法、胰酶法所获取的脊髓神经元产量更大、生长状态更好，可作为体外研究脊髓神经元病变的细胞模型。

英文摘要:

  【Abstract】 Objectives: To introduce a method to improve the culture technique of embryonic spinal cord neurons. Methods: 36 male and 36 female SD rats of childbearing age were mated and the female rats were pregnant. Firstly, six SD pregnant rats were selected respectively from each of the three groups of gestational ages of 10-12d, 13-15d, and 16-18d, and embryonic spinal cord were dissected and separated. Single cell suspension was prepared by routine trypsin digestion method. The total number of cells and the number of blue cells were counted under the microscope by cell counting plate and trypan blue staining, and the cell survival rate was calculated to determine the optimal gestational age of the material. Then, taking the above optimal gestational age as the sampling time point, six pregnant rats in each group prepared single cell suspension by three different digestion methods: enzyme free digestion, trypsin digestion and Accutase digestion. Using cell counting plate and trypan blue staining to count the total number of cells and blue cells under the microscope and calculate the cell survival rate. The isolated cells were inoculated, purified and cultivated for 1, 3, and 7 days. The growth vitality of the spinal neurons was observed under an inverted phase contrast microscope. The purity of neurons was identified and determined by double staining with the neuron-specific marker β-tubulin Ⅲ and nuclear marker. Results: The total number of cells and the number of viable cells in the 13-15d group were higher than those in the 10-12d group, and the cell survival rate in the 13-15d group was higher than that in the 16-18d group(P<0.05). The total number of cells, viable cells and cell survival rate in the Accutase group were significantly higher than those in the enzyme free group and the trypsin group(P<0.05). After culturing 7 days, the light ring gradually appeared around the neuron body and the neurites gradually extended to form a dense network under inverted phase contrast microscope. Compared with the enzyme free group and the trypsin group, the network of spinal cord neurons separated by the accutase enzyme method was more uniform and denser. After β tubulin Ⅲ and hoechst33342 double fluorescence staining identification, the purity of spinal cord neurons isolated and cultured in the Accutase group, enzyme free group and trypsin group was (90.29±2.55)%, (83.51±6.20)% and (88.10±4.07)% respectively with no significant difference(P>0.05). Conclusions: The best time for primary culture of rat spinal cord neurons was 13-15 days after pregnancy. Compared with conventional enzyme free and trypsin free methods, Accutase enzyme method can obtain more spinal cord neurons with better growth status, providing a good cell model for the study of spinal cord neuropathy in vitro.