Shimohama S, Ogawa N, Tamura Y, Akaike A, Tsukahara T, Iwata H, Kimura J
Department of Neurology, Faculty of Medicine, Kyoto University, Japan.
Brain Res. 1993 Dec 31;632(1-2):296-302. doi: 10.1016/0006-8993(93)91164-n.
The effect of recombinant human nerve growth factor (hNGF) and mouse NGF on cultured rat cortical neurons was examined. The DNA fragment coding the human NGF gene was isolated and inserted downstream from the SV40 promoter in a plasmid containing the dihydrofolate reductase cDNA, and this plasmid was introduced into Chinese hamster ovary (CHO) cells to establish cells producing recombinant hNGF. The recombinant hNGF protein secreted by CHO cells was confirmed to be biologically active in an assay using PC12 cells. Brief exposure of cortical cells to glutamate followed by incubation with glutamate-free medium reduced cell viability by 60-70% when compared with the control culture. Simultaneous addition of recombinant hNGF or mouse NGF to rat cortical cultures with glutamate did not affect this reduction of cell viability. However, 24 h pretreatment of rat cortical cultures with recombinant hNGF or mouse NGF resulted in a significant reduction of glutamate-induced neuronal damage. Mouse NGF also protected cortical neurons against N-methyl-D-aspartate (NMDA)- and kainate-induced neuronal damage. These findings suggest that NGF can protect cortical neurons against glutamate-induced neurotoxicity.
研究了重组人神经生长因子（hNGF）和小鼠神经生长因子对培养的大鼠皮质神经元的作用。分离编码人NGF基因的DNA片段，并将其插入含有二氢叶酸还原酶cDNA的质粒中SV40启动子的下游，然后将该质粒导入中国仓鼠卵巢（CHO）细胞以建立产生重组hNGF的细胞。在使用PC12细胞的测定中证实CHO细胞分泌的重组hNGF蛋白具有生物活性。与对照培养相比，将皮质细胞短暂暴露于谷氨酸，然后在无谷氨酸的培养基中孵育，细胞活力降低60 - 70%。在含有谷氨酸的大鼠皮质培养物中同时添加重组hNGF或小鼠NGF并不影响细胞活力的这种降低。然而，用重组hNGF或小鼠NGF对大鼠皮质培养物进行24小时预处理导致谷氨酸诱导的神经元损伤显著降低。小鼠NGF还可保护皮质神经元免受N-甲基-D-天冬氨酸（NMDA）和红藻氨酸诱导的神经元损伤。这些发现表明NGF可以保护皮质神经元免受谷氨酸诱导的神经毒性。
