Tonoki H
Department of Pediatrics, Hokkaido University School of Medicine, Sapporo, Japan.
Hokkaido Igaku Zasshi. 1994 Sep;69(5):1261-74.
Glutathione reductase (GR) protects tissues from oxidant injury by catalysing the reduction of glutathione disulfide (GSSG) to glutathione (GSH). In order to study the effect of GR in protecting cells from oxidant injury, we generated Chinese hamster ovary (CHO) cell lines stably transformed after antisense-oriented gene transfection. The coding region of the human GR was cloned using revere transcription PCR method and selected by transient expression study in mammalian cells. A clone HGR135 showed overexpression of GR in CHO cells and was proved to have no base substitution. This clone, then, was ligated into MEP4 expression vector in an antisense orientation to the human metallothionein promoter and transfected to CHO cells with polybrene. Among 12 cell lines isolated, G17 showed to have the least GR activity (48% of the control), while another four were mildly GR deficient. Southern hybridization of genomic DNA digests and transformation experiment on E. coli revealed that the promoter-antisense coding region component was integrated. Northern hybridization detected reduced amount of GR transcript but no antisense message. Baseline cellular GSH concentrations were lower in G17 than in control (25.7 +/- 2.5 vs. 36.1 +/- 1.9 nmole/mg protein, P < 0.05), while cellular GSSG concentrations were higher (0.61 +/- 0.19 vs. 0.39 +/- 0.09 nmole/mg protein, P < 0.05). After four hours of treatment of G17 and control cells with increasing doses (1 to 10 mM) of t-butylhydroperoxide (t-BuOOH), cellular GSH concentrations in G17 decreased with an elevation of GSSG concentration at 1 mM followed by no further increase at higher t-BuOOH concentration, while GSSG concentrations increased in the control cells without reduction of GSH concentrations at 1-5 mM t-BuOOH treatment. The concentrations of GSH were lower in G17 than in controls at all doses of t-BuOOH. Four hours of exposure to 10 mM t-BuOOH resulted in greater LDH release in G17 than in control (57.3 +/- 4.7 vs. 32.1 +/- 6.5%, P < 0.05). Similarly, G17 cells released more of their LDH to the media than did CHO cells in response to exposure to 95% O2 for 72 hours (19.3 +/- 5.9 vs. 11.9 +/- 5.4%, P < 0.05). The partial GR deficiency in G17 cells impairs their ability to recycle GSSG and this deficiency offers the best explanation for the increased sensitivity of these cells to injury by t-BuOOH or hyperoxia.
谷胱甘肽还原酶（GR）通过催化谷胱甘肽二硫化物（GSSG）还原为谷胱甘肽（GSH）来保护组织免受氧化损伤。为了研究GR在保护细胞免受氧化损伤中的作用，我们通过反义定向基因转染生成了稳定转化的中国仓鼠卵巢（CHO）细胞系。使用逆转录PCR方法克隆人GR的编码区，并通过在哺乳动物细胞中的瞬时表达研究进行筛选。一个克隆HGR135在CHO细胞中显示GR过表达，并且被证明没有碱基替换。然后，将该克隆以与人类金属硫蛋白启动子反义的方向连接到MEP4表达载体中，并用聚凝胺转染到CHO细胞中。在分离出的12个细胞系中，G17的GR活性最低（为对照的48%），而另外四个细胞系的GR活性轻度缺乏。基因组DNA消化产物的Southern杂交和大肠杆菌转化实验表明启动子-反义编码区成分已整合。Northern杂交检测到GR转录本数量减少，但没有反义信息。G17细胞中的基线细胞内GSH浓度低于对照（25.7±2.5对36.1±1.9纳摩尔/毫克蛋白质，P<0.05），而细胞内GSSG浓度较高（0.61±0.19对0.39±0.09纳摩尔/毫克蛋白质，P<0.05）。用递增剂量（1至10 mM）的叔丁基过氧化氢（t-BuOOH）处理G17和对照细胞4小时后，G17细胞中的细胞内GSH浓度在1 mM时随着GSSG浓度升高而降低，在较高t-BuOOH浓度下不再进一步增加，而在1-5 mM t-BuOOH处理时，对照细胞中的GSSG浓度增加而GSH浓度没有降低。在所有t-BuOOH剂量下，G17细胞中的GSH浓度均低于对照。暴露于10 mM t-BuOOH 4小时导致G17细胞中乳酸脱氢酶（LDH）释放量比对照细胞更多（57.3±4.7对32.1±6.5%，P<0.05）。同样，暴露于95%氧气72小时后，G17细胞向培养基中释放的LDH比CHO细胞更多（19.3±5.9对11.9±5.4%，P<0.05）。G17细胞中部分GR缺乏损害了它们回收GSSG的能力，这种缺乏为这些细胞对t-BuOOH或高氧损伤敏感性增加提供了最佳解释。
