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目的:探讨建立简单、稳定的大鼠海马神经元体外原代培养方法。方法:分离出生24 hr大鼠海马组织,采用机械吹打法或胰蛋白酶消化法制备单细胞悬液,以含10%胎牛血清的DMEM/F12培养基接种,24 hr后更换含2%B27的DMEM/F12无血清培养基饲养,每3 d全量换液,观察神经元形态,培养第7d检测神经元纯度,MTT法测定神经细胞活性,比较两种操作方法的培养效果。结果:机械吹打法可缩短实验操作时间,减少培养污染率,降低了操作难度,培养出的神经元纯度高、活性好。结论:机械吹打法操作简便,结果稳定,是大鼠海马神经元体外原代培养的好方法。
Objective: To explore a simple and stable rat primary culture method of hippocampal neurons in vitro. Methods: The hippocampus tissue of 24 h was isolated and single cell suspension was prepared by mechanical pipetting or trypsin digestion. The cells were inoculated with DMEM / F12 medium containing 10% fetal bovine serum and replaced with 2% B27 The DMEM / F12 serum-free medium was fed every 3 d. The morphological changes of neurons were observed. The purity of neurons was detected on the 7th day. The activity of neurons was measured by MTT assay. The culture effects of the two methods were compared. Results: The mechanical blowing method can shorten the experimental operation time, reduce the culture pollution rate and reduce the operation difficulty. The cultured neurons have high purity and good activity. Conclusion: The mechanical blowing method is simple and stable, which is a good method for primary culture of rat hippocampal neurons in vitro.