目的：建立科学简易的大鼠皮质神经元的体外培养方法，观察尿激酶对其干预后的表现。方法：取24 h 内新生Wistar 大鼠脑皮质，用浓度为2 mg/mL 的木瓜酶和适量DNase I 酶共同消化，并用配置好的无血清 Neurobasal 培养基接种培养，6 d 后免疫荧光法鉴定神经元纯度；分别配置含尿激酶终浓度为5 000 U/mL、 8 000 U/mL、10 000 U/mL、15 000 U/mL和20 000 U/mL的Neurobasal 培养基并进行全量换液，镜下观察不同 浓度尿激酶干预后神经元的变化。结果：培养6 d，神经元分化成熟，胞质丰富，树突及轴突舒展延长，可见密 集的神经纤维网络，免疫荧光鉴定神经元纯度为88.2%；尿激酶干预后，发现尿激酶浓度在5 000~10 000 U/mL 时，2 h 内镜下观察培养体系无明显变化，但随时间的延长，尿激酶浓度为10 000 U/mL作用4 h 时，可见少量 神经元细胞破碎崩解，细胞间网状结构减少。尿激酶浓度在15 000 U/mL 及20 000 U/mL 时，干预2 h 发现 神经元细胞崩解，网状结构消失。结论：本实验建立了一种简易、高效的体外培养新生大鼠皮质神经元的方 法，尿激酶浓度在5 000~10 000 U/mL时，2 h 内对体外神经元培养体系是安全的。

To establish a more scientific and simple method for the culturing of cerebral cortical neurons from newborn rats and observe the interventional effect of different concentrations of urokinase. Methods: The cerebral cortex of newborn Wistar rats was extracted and then digested by papaya enzyme (2 mg/ mL) and an appropriate amount of DNase I enzyme. Neurons were cultured with serum-free Neurobasal medium. After 6 days, the purity of neurons was identified by immunofluorescence. Neurobasal media with final urokinase concentrations of 5 000 U/mL, 8 000 U/mL, 10 000 U/mL, 15 000 U/mL, and 20 000 U/mL were prepared for media changes. Neurons were observed under microscope for changes due to intervention from various concentrations of urokinase. Results: After 6 days of culture, the neurons were well-differentiated with abundant cytoplasm, and neuronal dendrites and axons extended and overlapped into a dense network. Neurons purity was identified as 88.2% by immunofluorescence staining. After intervention with urokinase at a concentration of 5 000 to 10 000 U/mL, no significant change in the culture system was observed under microscope within 2 h, but over time, a small number of neuronal cell disruption and network structure decrease could be observed with 10 000 U/mL at 4 h. With 15 000 U/mL and 20 000 U/mL, at 2 h after the intervention, fragmented material was observed at the bottom of the medium, and the intercellular network structure disappeared. Conclusion: In this study, a simple and efficient method of culturing newborn rat cortical neurons was established. When urokinase concentration was between 5 000 and 10 000 U/mL, it was safe to culture neurons within 2 h in vitro.