大鼠皮质神经元的体外培养及不同浓度尿激酶干预后的观察

摘要

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.%Objective: 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 invitro.