一种改良的原代神经元牵张损伤模型的构建与评估

Construction and evaluation of a modified stretch injury model of primary cultured neurons

目的探讨原代神经元牵张损伤模型的改良方法及其可行性。方法提取出生24 h内SD乳鼠皮层组织进行原代神经元培养,利用智能真空负压机和弹力膜培养板对经典的神经元牵张损伤模型(CICⅡ模型)进行改良,根据弹力膜形变距离将细胞分为对照组(只种植细胞,不做牵张损伤)、轻度损伤组(形变距离7 mm)、中度损伤组(形变距离10 mm)和重度损伤组(形变距离13 mm)。显微镜观察细胞形态变化,NeuN免疫荧光染色鉴定细胞纯度;免疫印迹法检测凋亡相关蛋白(cleaved Caspase-3、Bcl-2)表达,比色法测定培养液乳酸脱氢酶(LDH)活性。结果原代神经元培养5 d,显微镜下观察发现细胞生长良好,NeuN染色显示神经元纯度在95%以上。中度损伤后即刻,显微镜下发现神经元轴索断裂,部分细胞呈空泡样变。与对照组相比,损伤组cleaved Caspase-3表达水平、细胞培养液LDH水平均明显上升(P<0.05),而Bcl-2表达水平明显下降(P<0.05)。损伤组cleaved Caspase-3表达水平、细胞培养液LDH水平与伤程度呈明显正相关(P<0.05),而Bcl-2表达水平与伤程度呈明显负相关(P<0.05)。结论新生大鼠皮层体外培养所获原代神经元纯化率较高。本研究改良模型可模拟神经元损伤,使用方便、可重复性好,适合颅脑损伤和细胞力学研究。

Objective To explore an improved method for establishing a primary neuronal stretch injury model and its feasibility.Methods Cortical tissues from SD rats within 24 hours of birth were extracted for primary neuronal culture.An improved method for the classic neuronal stretch injury model(CICⅡmodel)was developed using an intelligent vacuum negative pressure machine and elastic membrane culture plate.Cells were divided into control group(no stretch injury),mild injury group(deformation distance 7 mm),moderate injury group(deformation distance 10 mm),and severe injury group(deformation distance 13 mm)based on the elastic membrane deformation distance.Cell morphology changes were observed under a microscope,and NeuN immunofluorescent staining was used to identify cell purity.Apoptosis-related proteins(cleaved Caspase-3,Bcl-2)expression was detected by immunoblotting,and lactate dehydrogenase(LDH)activity in the culture medium was measured using a colorimetric assay.Results On the 5th day of cell culture,cells showed good growth under the microscope,with NeuN staining indicating neuronal purity above 95%.Immediately after moderate injury,axonal rupture and vacuolated cell morphology were observed under the microscope.Compared to the control group,the expression levels of cleaved Caspase-3 and LDH in the culture medium were significantly increased(P<0.05),while Bcl-2 expression was significantly decreased(P<0.05)in the injury group.The expression levels of cleaved Caspase-3 and LDH in the culture medium were positively correlated with the degree of injury(P<0.05),while Bcl-2 expression was negatively correlated with the degree of injury(P<0.05).Conclusions High purity of primary neurons was obtained from cultured cortical tissues of newborn rats.The improved model in this study can simulate neuronal injury,is convenient to use,and has good reproducibility,making it suitable for research on cranial injuries and cell mechanics.