SCRENING OF IN NEW-BORN RAT LUNG GENES RESPONDING TO OXYGEN TENSION SHIFT

新生大鼠肺中响应氧张力变化的基因的筛选

• 批准号: 06807068
• 负责人: TAKAHASHI Yuji
• 金额: $ 1.28万
• 依托单位: Tokyo University of Pharmacy and Life Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06807068/
• 关键词: HYPOXIA; REOXYGENATION; DIFFERENTIAL DISPLAY; PROLYL 4-HYDROXYLASE; PHOSPHOGLYCERATE MUTASE; RAT; differential display; Prolyl hydroxylase; 酸素刺激; ラット; 新生仔; 肺; RT-PCR; Differential Display

At birth, mammals are exposed to oxygen tension shift from low O2 in the uterus (PO2 is 12 Torr) to high O2 in atmospheric environment (PO2 is more than 100 Torr). Especially, the lung epithelium receives drastic increase in oxygen tension at the beginning of respiration. In this study, we screened the genes which respond to oxygen tension shift using the fluorescent differential display method in order to explore the adaptation mechanism of mammals to oxygen tension shift.Fibroblast isolated from fetal rat lung and SV40 immortalized type 2 alveolar epithelial cell were cultured at various oxygen tension. Total RNA was isolated from these cells before and after exposures to hypoxia. After RT-PCR amplification with FITC-labeled anchor primers and arbitrary primers, DNA fragments were separated by 6% PAGE and analyzed by Pharmacia Fragment Manager or autoradiography.Four DNA fragments were differentially expressed by hypoxia and three of them have the sequence identical to 3' end of aldolase a, phosphoglycerate mutase type B subunit and prolyl 4-hydroxylase. The last DNA fragment showed no homology to any genes in data bank.Prolyl 4-hydroxylase was up-regulated by hypoxia and down-regulated by oxygen tension shift from hypoxia to normoxia. Furthermore, the RNA was more abundant in the fetal lung than in the adult lung. These data suggest that collagen metabolism may be regulated by oxygen tension.

哺乳动物出生时所面临的氧张力从子宫内的低O2（PO2为12托）转变为大气环境中的高O2（PO2超过100托）。特别是，在呼吸开始时，肺上皮的氧张力急剧增加。本研究采用荧光差异显示法筛选响应氧张力变化的基因，以探讨哺乳动物对氧张力变化的适应机制。培养胎鼠肺成纤维细胞和SV40永生化2型肺泡上皮细胞在不同的氧张力下。在暴露于缺氧之前和之后，从这些细胞中分离出总RNA。用 FITC 标记的锚定引物和任意引物进行 RT-PCR 扩增后，通过 6% PAGE 分离 DNA 片段，并通过 Pharmacia Fragment Manager 或放射自显影进行分析。 4 个 DNA 片段因缺氧而差异表达，其中 3 个片段与 3 个序列相同' 醛缩酶 A、磷酸甘油酸变位酶 B 型亚基和脯氨酰 4-羟化酶的末端。最后的DNA片段与数据库中的任何基因均无同源性。脯氨酰4-羟化酶在缺氧时上调，在氧张力从缺氧转为常氧时下调。此外，胎儿肺中的RNA比成人肺中的RNA更丰富。这些数据表明胶原蛋白代谢可能受到氧张力的调节。

项目成果

Martin Joyce-Brady: "Synthesis and Release of Amphypathic α-Glutamyl" The Journal of Biological Chemistry. 269. 14219-14226 (1994)

Martin Joyce-Brady：“两亲性 α-谷氨酰的合成和释放”《生物化学杂志》269。14219-14226 (1994)

Yuji Takahashi: "Cytochrome P4502B1 Immunoreactivity in Bronchiolar" Toxicology and Applied Pharmacology. 128. 207-215 (1994)

Yuji Takahashi：“细支气管中的细胞色素 P4502B1 免疫反应性”毒理学和应用药理学。