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目的:观察组织型纤溶酶原激活物(tPA)对体外不同条件培养的神经元的损伤作用,探讨神经源性丝氨酸蛋白酶抑制剂(NSP)是否可以减轻tPA对神经元的损伤。方法:体外原代培养大鼠皮质神经元,建立缺氧复氧(H/R)模型。在不同的培养条件下使用不同的试剂干预方法。采用倒置相差显微镜观察免疫荧光染色后的细胞形态学变化、活细胞计数试剂盒测定神经元存活率、LDH释放实验测定细胞毒性和Tunel试剂盒测定细胞凋亡率。结果:tPA对正常神经元具有毒性损伤作用。经tPA干预的H/R组与单纯H/R组相比,前者神经元损伤加重(P<0.05)。NSP+tPA联合干预的H/R组细胞损伤均轻于tPA干预的H/R组(P<0.05)。结论:NSP可减轻tPA对神经元的毒性损伤而起到保护神经元的作用。
OBJECTIVE: To observe the effect of tissue plasminogen activator (tPA) on neurons cultured in vitro under different conditions and to explore whether neurogenic serine protease inhibitor (NSP) can reduce the damage of neurons by tPA. Methods: Primary cortical neurons were cultured in vitro and hypoxia-reoxygenation (H / R) model was established. Different reagent interventions are used under different culture conditions. The morphological changes of cells after immunofluorescence staining were observed by inverted phase contrast microscope. The viability of neurons was measured by viable cell counting kit, the cytotoxicity by LDH release assay and the apoptosis rate by Tunel kit. Results: tPA had a toxic effect on normal neurons. Compared with the H / R group, the neuronal injury was increased in the H / R group (P <0.05). The cell injury of H / R group treated with NSP + tPA was lighter than that of H / R treated with tPA (P <0.05). Conclusion: NSP can reduce neurotoxicity of tPA and protect neurons.