CAJ | 학술논문

目的：探讨建立较为简便的新生大鼠皮层神经元细胞体外无血清原代培养方法。方法取新生大鼠（24 h ）大脑皮层组织，消化后种植在有多聚-L-赖氨酸包被的六孔培养板中，以含10％胎牛血清DEM E-HG培养液种植，4～8 h后换用含B27的Neurobasal培养基维持饲养。于不同时间在倒置相差显微镜下观察形态变化，分别采用RT-PCR、Western blot、免疫组织化学对神经元特异性标记物神经元特异性烯醇化酶（NSE）基因及蛋白进行鉴定。结果2～8 h神经元细胞贴壁，随着时间延长，形态多变，突起逐渐增多，神经元突起间相互接触形成网络，培养7～10 d神经元胞体最为丰满，通过RT-PCR、Western blot和免疫组织化学证明所分离培养的是神经元细胞。结论该方法简单易行，神经元纯度较高，可作为神经元体外培养的良好模型用于以后的研究。
목적：탐토건립교위간편적신생대서피층신경원세포체외무혈청원대배양방법。방법취신생대서（24 h ）대뇌피층조직，소화후충식재유다취-L-뢰안산포피적륙공배양판중，이함10％태우혈청DEM E-HG배양액충식，4～8 h후환용함B27적Neurobasal배양기유지사양。우불동시간재도치상차현미경하관찰형태변화，분별채용RT-PCR、Western blot、면역조직화학대신경원특이성표기물신경원특이성희순화매（NSE）기인급단백진행감정。결과2～8 h신경원세포첩벽，수착시간연장，형태다변，돌기축점증다，신경원돌기간상호접촉형성망락，배양7～10 d신경원포체최위봉만，통과RT-PCR、Western blot화면역조직화학증명소분리배양적시신경원세포。결론해방법간단역행，신경원순도교고，가작위신경원체외배양적량호모형용우이후적연구。
Objective To establish a serum-free primary culture method for cortical neurons of new-born rats .Methods The cortical tissue was digested and the cells were planted in the medium containing 10% fetal bovine serum ,and then maintained feed-ing with neurobasal medium containing B27 after 4 to 8 h .The morphology was observed under phase-contrast microscope .RT-PCR ,Western blot and immunocytochemistry were applied to identify the expression of NSE gene and protein in neurons .Results A large number of neurons began to adhere to the cover glasses after 2 to 8 h .They showed different shapes-shuttle ,triangle pyram-idal ,or no regular after clinging to the plate .Their processes connected to nets and were different in length and thickness .They well developed at the 7th to 10th day .The isolated and cultured cells were confirmed as neurons by RT-PCR ,Western blot and immuno-cytochemistry .Conclusion This technique is an easy and practical tool for primary culture of new-born rats cortical neurons with high purity ,and can be used as an in-vitro model of research .