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目的:观察体外培养的大鼠大脑皮层神经元在模拟脑缺血再灌注后的变化以及中药抗呆Ⅰ号的影响。方法:先对分离纯化培养的大脑皮层神经元进行体外模拟脑缺血再灌注损伤模型的建立,然后采用MTT法测定神经元的活性和存活率,用台盼蓝染色法测定其死亡率,用比色法测定乳酸脱氢酶(LDH)的漏出率,用组化染色法测定一氧化氮合成酶(NOS)的活性。结果:体外培养的大鼠大脑皮层神经元随缺血和再灌注时间的延长细胞的活性和存活率逐渐下降、死亡率逐渐升高、培养液中LDH的漏出率逐渐升高、细胞NOS表达强阳性细胞数在缺血4h(34.6±3.847)和再灌3h(20.6±3.362)时显著增高。模型组与正常组相比,差异有非常显著性意义(t=13.236,8.038,P<0.01)。抗呆Ⅰ号可影响其上述指标的变化。结论:抗呆Ⅰ号可能通过防止氧化磷酸化脱耦联而保护线粒体、防止脂质过氧化及通透性增加而保护细胞膜、抑制NOS活性的反应性增强而防止一氧化氮(NO)及其衍生的毒性自由基的损伤等途径而发挥对神经元的保护作用。
OBJECTIVE: To observe the changes of rat cerebral cortical neurons cultured in vitro after simulated cerebral ischemia-reperfusion and the effects of traditional Chinese medicine Anti-deafness I. METHODS: The isolated cerebral cortex neurons were cultured in vitro to establish a model of cerebral ischemia-reperfusion injury. The activity and survival rate of neurons were measured by MTT assay, and the mortality was determined by trypan blue staining. The colorimetric method was used to determine the leakage rate of lactate dehydrogenase (LDH), and the activity of nitric oxide synthase (NOS) was determined by histochemical staining. RESULTS: In vitro, the activity and survival rate of neurons in rat cerebral cortical neurons gradually decreased with the prolongation of ischemia and reperfusion, the mortality rate gradually increased, the leakage rate of LDH in culture medium gradually increased, and the expression of NOS in cells was strong. The number of positive cells was significantly increased at 4 h (34.6±3.847) and 3 h (20.6±3.362) reperfusion. The difference between the model group and the normal group was significant (t=13.236, 8.038, P<0.01). Anti-dead I can affect the changes in the above indicators. Conclusion: Kangde I may protect against the mitochondria by preventing the dephosphorylation of oxidative phosphorylation, prevent the lipid peroxidation and increase the permeability, protect the cell membrane, inhibit the increase of the reactivity of NOS activity and prevent nitric oxide (NO) and its Detoxification of toxic free radicals, etc., plays a protective role against neurons.