Physiological significance of pyruvate kinase isozymes in erythrocyte.

红细胞中丙酮酸激酶同工酶的生理意义。

• 批准号: 11670153
• 负责人: KANNO Hitoshi
• 金额: $ 2.3万
• 依托单位: NIHON UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670153/
• 关键词: hemolytic anemia; apoptosis; glycolysis; reactive oxygen species; disease model animals; Friend cells; 遺伝子治療; トランスジェニックマウス; hemolytic anemia; apoptosis; glycolysis; reactive oxygen species; disease model animals; Friend cells; 遺伝子治療; トランスジェニックマウス

Pyruvate kinase (PK) is a rate-limiting enzyme of the glycolytic pathway and PK deficiency is one of the most prevalent causes of hereditary non-spherocytic hemolytic anemia. The Pk-1^<slc> mouse is found to be a spontaneous mutant of the murine LR-type PK gene, and manifests hemolytic anemia and splenomegaly. We showed that apoptotic cells were notably increased in spleen of the Pk-1^<slc> by the TUNEL method, and that the erythroid-specific expression of normal PK gene ameliorated apoptosis in the genetic rescue experiment. Flow cytometric analysis showed that Annexin V / Ter119-double positive cells were significantly increased in the Pk-1slc mouse compared to wild type CBA mice, confirming apoptotic cells were of erythroid lineage. In this study, we examined a physiological role of glycolysis upon apoptosis of the erythroid cells using two Friend erythroleukemic cells, SLC and CBA.These cell lines have been establushed from the Pk-1^<slc> and a wild-type CBA mouse, respectively. As previously reported, apoptosis spontaneously occurred in the Friend cells and was inducible in both cells by glucose deprivation or 0.1-20mM 2-deoxyglucose (2DG). SLC cells were more susceptible to apoptosis by either shorter exposure to glucose deprivation or lower 2DG concentration. The detailed mechanism are still unclear, but we have shown that reactive oxygen species (ROS) might be accountable for these apoptotic changes.

丙酮酸激酶（PK）是糖酵解途径的速率限制酶，而PK缺乏症是遗传性非细胞性溶血性溶血性贫血的最普遍原因之一。发现PK-1^<slc>小鼠是鼠LR型PK基因的自发突变体，并表现出溶血性贫血和脾肿大。我们表明，通过TUNEL方法，PK-1^<slc>的脾脏凋亡细胞显着增加，并且在遗传救援实验中，正常PK基因的红细胞特异性表达改善了凋亡。流式细胞仪分析表明，与野生型CBA小鼠相比，PK-1SLC小鼠的膜联蛋白V / TER119双阳性细胞显着增加，确认凋亡细胞为红细胞性谱系。在这项研究中，我们研究了使用两个朋友红血病细胞，SLC和CBA检查糖酵解对红斑细胞凋亡的生理作用。这些细胞系分别是从PK-1^<slc>和一个野生型CBA小鼠中建立的。如前所述，凋亡自发发生在朋友细胞中，并通过葡萄糖剥夺或0.1-20mm 2-脱氧葡萄糖（2DG）在两个细胞中诱导。 SLC细胞更容易通过较短的葡萄糖剥夺或较低的2DG浓度来凋亡。详细的机制仍不清楚，但我们已经表明，活性氧（ROS）可能对这些凋亡变化负责。