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Analysis of cell signaling of Glia Maturation Factor -beta and -gamma in the central nervous system

中枢神经系统神经胶质成熟因子-β和-γ的细胞信号传导分析

基本信息

批准号：
15591121
负责人：
ASAI Kiyofumi
金额：
$ 2.24万
依托单位：
NAGOYA CITY UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591121/
关键词：
Glia GMFB GMFG Astrocyte EIA

项目摘要

1, Analysis of promoter activity of the human glia maturation factor-gamma geneWe determined the organization of the 9.5-kb hGMFG gene and characterized its promoter activity. The 5'-flanking region of the first exon has no TATA or CAAT boxes within a 226-bp sequence upstream from the initiation codon. Primer extension analysis and 5'RACE (rapid amplification of cDNA 5' ends) identified multiple transcription initiation sites within the region -84 to -70 nucleotides from the first ATG codon in a Kozak consensus sequence. A core promoter region was determined by transfecting a series of deletion constructs with a dual luciferase reporter system into rat astrocyte-derived ACT-57 cells.2, Development of two-site enzyme immunoassays (EIA) for glia maturation factor beta (GMFB) and gamma (GMFG)We developed sensitive and specific two-site enzyme immunoassays (EIA) for glia maturation factor beta (GMFB) and gamma (GMFG) using specific antibodies raised in rabbits. These assay systems enabled … More us to identify GMFB and GMFG (GMFs) in both human and rat samples and they were used to investigate the tissue distribution and serum concentrations of human and rat GMFs. In the case of rat, relatively high levels of GMFB were found in the central nervous system, except for the spinal cord, and in thymus and colon. Higher levels of GMFG were found in the thymus, spleen and colon. The distribution of GMFs in human was similar to that in rat. In the rat, the maximum serum concentration of GMFG was at 4 weeks of age. The decrease in its level was rapid for the first 30 days of life in both sexes. On the other hand, the concentration of GMFB in serum did not change significantly with age. Similarly, in human, the concentration of GMFG in serum was highest in the 21-30-year-old group and began to decrease rapidly in the 30-year-old group. In contrast, the concentration of GMFB did not change significantly during this period. No significant sex differences in the serum levels of GMFs were observed in human and rat.3, Analysis of expression of glia maturation factor beta after cryogenic injuryWe investigated the expression of GMFB during 56 days after cryogenic brain injury, using immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and enzyme immunoassay. Immunohistochemical analysis demonstrated that the GFAP-positive astrocytes around the lesion expressed GMFB protein, peaking 14 days after injury. Weak astrocytic expression of GMFB immunoreactivity was seen in sham-operated animal brains. Cryogenic injury (CI) induced GMFB mRNA in the lesioned side after 7 days with a maximum at 14 days. Western blotting revealed the induction of GMFB protein starting 1 day after injury, and continuing until 14 days after injury. In the enzyme immunoassay, GMFB protein concentration peaked 14 days after injury in extracts from the injured side of the brain, whereas in serum it peaked 1 day after injury. These data indicate that the expression of GMFB increased in the astrocytes around the lesioned area after cortical cryogenic brain injury. Less

1、人胶质成熟因子-γ基因启动子活性分析我们测定了9.5kb hGMFG基因的结构，并对其启动子活性进行了分析。第一外显子的5 '侧翼区在起始密码子上游的226-bp序列内没有TATA或CAAT盒。引物延伸分析和5 'RACE（cDNA 5'末端快速扩增）鉴定了Kozak共有序列中第一个ATG密码子的-84至-70个核苷酸区域内的多个转录起始位点。通过将一系列具有双荧光素酶报告系统的缺失构建体转染到大鼠星形胶质细胞衍生的ACT-57细胞中来确定核心启动子区。胶质细胞成熟因子β（GMFB）和γ（GMFG）双位点酶免疫分析法的建立我们建立了敏感、特异的胶质细胞成熟因子β（GMFB）和γ（GMFG）双位点酶免疫分析法用的是从兔子身上提取的特异性抗体。这些分析系统能够 ...更多信息我们鉴定了人和大鼠样品中的GMFB和GMFG（GMFs），并将其用于研究人和大鼠GMFs的组织分布和血清浓度。在大鼠的情况下，在中枢神经系统（脊髓除外）以及胸腺和结肠中发现相对高水平的GMFB。在胸腺、脾脏和结肠中发现较高水平的GMFG。GMFs在人体内的分布与大鼠相似。在大鼠中，GMFG的最大血清浓度是在4周龄。在出生后的前30天，两性的血肌酐水平都迅速下降。另一方面，血清中GMFB浓度随年龄变化不显著。同样，在人体中，血清中GMFG的浓度在21-30岁组中最高，并在30岁组中开始迅速下降。相反，GMFB的浓度在此期间没有显著变化。应用免疫组化、逆转录聚合酶链反应（RT-PCR）、Western blotting和酶免疫分析等方法检测了冷冻性脑损伤后56天内GMFB的表达。免疫组化结果显示，损伤周围的GFAP阳性星形胶质细胞表达GMFB蛋白，在损伤后14天达到高峰。在假手术动物脑中可见GMFB免疫反应性的弱星形胶质细胞表达。低温损伤（CI）后7 d损伤侧GMFB mRNA表达增加，14 d达高峰。Western blotting显示GMFB蛋白在伤后1天开始诱导表达，并持续至伤后14天。在酶免疫分析中，GMFB蛋白浓度在损伤后14天达到峰值，而在血清中，它在损伤后1天达到峰值。这些数据表明，GMFB的表达增加，在皮质冷冻脑损伤后的损害区周围的星形胶质细胞。少