CAJ | 학술논문

目的：建立高纯度的新生SD大鼠皮质神经元原代培养方法。方法：取24 h内的新生SD大鼠皮质，用木瓜酶和DNaseⅠ共同消化，5%胎牛血清终止消化，吹打分离组织获得单细胞悬液，进行细胞计数，用无血清DMEM/F12种植培养，4 h后换成用无血清Neurobasal配制的维持培养液继续培养，尼氏小体染色和免疫荧光法鉴定神经元的纯度。结果：培养第10 d，神经元胞体饱满，结构清晰完整，光晕明显，折光性强，可见粗长的树突和轴突，相邻细胞形成紧密网状联系，神经元纯度达到96%以上。结论：经改良和优化，无须添加阿糖胞苷抑制胶质细胞的生长即能够获得生长状态良好、高纯度的神经元。
목적：건립고순도적신생SD대서피질신경원원대배양방법。방법：취24 h내적신생SD대서피질，용목과매화DNaseⅠ공동소화，5%태우혈청종지소화，취타분리조직획득단세포현액，진행세포계수，용무혈청DMEM/F12충식배양，4 h후환성용무혈청Neurobasal배제적유지배양액계속배양，니씨소체염색화면역형광법감정신경원적순도。결과：배양제10 d，신경원포체포만，결구청석완정，광훈명현，절광성강，가견조장적수돌화축돌，상린세포형성긴밀망상련계，신경원순도체도96%이상。결론：경개량화우화，무수첨가아당포감억제효질세포적생장즉능구획득생장상태량호、고순도적신경원。
Objective: To establish a method for cultivation of cerebral cortex neuronal cells of newborn SD rat. Methods: The cortexes from newborn(less than 24 h) SD rat were obtained and digested by 2 mg/mL papain and appropriate amount of DNaseⅠ, terminated digestion with terminating medium and then dissociated into single cell suspension, counted and plated with plating medium. After cultivated in plating medium for 4 h, the neuronal cells were cultured in maintenance medium containing Neurobasal medium, B27 and glutamine. The Nissl's stain?ing and indirect immunofluorescence were used to indicate neuronal cells. Results: The neuronal cells on 10th day showed that the body of neuronal cells were full, the structure of neuronal cells were clear and integrity, the axons and the dendrites were thick and long,adjacent cells formed a tight mesh contact. As indicated by indirect immunofluorescence using antibodies against neuron specific tubulin βⅢ and Nissl's staining, the purity of the neu?ronal cultures was above 96%. Conclusion: This simplified method(added DNaseⅠ, serum-free medium and cyto?sine-arabinofuranoside-free) is cost-effective for primary culture of cerebral cortex neuronal cells and the neurons obtained showed high uniformity, purity and long-term viability.