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，暴露在从宫内低氧(PO2为12托)到大气环境中高氧(PO2大于100托)的氧分压变化中。尤其是在呼吸开始时，肺上皮细胞的氧分压急剧增加。在本研究中，我们利用荧光差异显示的方法筛选了响应氧压变化的基因，以探索哺乳动物对氧压变化的适应机制。从胎鼠肺分离出的成纤维细胞和SV40永生化的2型肺泡上皮细胞在不同的氧分压下培养。从这些细胞中提取缺氧前后的总RNA。用FITC标记的锚定引物和随机引物进行RT-PCR扩增后，用6%PAGE分离DNA片段，用Pharmacia Fragment Manager或放射自显影进行分析，其中4个DNA片段在低氧条件下有差异表达，其中3个片段的序列与醛缩酶A的3‘端、磷酸甘油变位酶B亚基和Pro-4-羟基酶的序列相同。最后一个DNA片段与数据库中的任何基因都没有同源性。Pro-4-羟基酶在低氧条件下上调，而在低氧至常氧状态下氧分压变化时下调。此外，胎儿肺中的RNA比成人肺中的更丰富。这些数据表明，胶原代谢可能受氧分压的调节。

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。