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

1、人胶质细胞成熟因子-γ基因启动子活性的分析我们确定了9.5-kb的hGMFG基因的组织结构，并对其启动子活性进行了鉴定。第一个外显子的5‘侧翼区在起始密码子上游226个碱基的序列中没有TATA或CAAT盒。引物延伸分析和5‘RACE(快速扩增cDNA5’末端)在Kozak共有序列中第一个ATG密码子的-84~-70个核苷酸区域内发现了多个转录起始点。用双荧光素酶报告系统将一系列缺失结构导入大鼠星形胶质细胞来源的ACT-57细胞，确定其核心启动子区域。2.胶质细胞成熟因子β(GMFB)和γ(GMFG)双位点酶免疫分析方法的建立利用兔体内的特异性抗体，建立了灵敏、特异的胶质细胞成熟因子β(GMFB)和γ(GMFG)双位点酶免疫分析方法。这些检测系统使…为了在人和大鼠标本中鉴定GMFB和GMFG(GMFF)，并用它们来研究人和大鼠GMFs的组织分布和血药浓度。在大鼠中，除脊髓外，中枢神经系统、胸腺和结肠中的GMFB水平相对较高。胸腺、脾和结肠中GMFG水平较高。GMFs在人体内的分布与大鼠相似。在大鼠中，GMFG的血药浓度在4周龄时最高。在生命的前30天，无论男女，它的水平都迅速下降。血清GMFB浓度随年龄变化不明显。同样，在人体中，21-30岁组血清中GMFG浓度最高，30岁组开始迅速下降。相比之下，GMFB的浓度在这段时间内没有明显变化。冷冻脑损伤后GMFB的表达分析采用免疫组织化学、逆转录聚合酶链式反应(RT-PCR)、Western blotting和酶免疫分析等方法，研究了冷冻脑损伤后56d内GMFB的表达。免疫组织化学分析显示，损伤周围GFAP阳性星形胶质细胞表达GMFB蛋白，伤后14天达高峰。假手术组动物脑内GMFB免疫反应阳性星形胶质细胞表达较弱。低温损伤后7d，损伤侧GMFB基因表达增强，14d达高峰。Western blotting显示GMFB蛋白从伤后1d开始表达，一直持续到伤后14d。在酶免疫分析中，损伤侧脑组织提取液中GMFB蛋白浓度在伤后14天达到峰值，而血清中GMFB蛋白浓度在伤后1天达到峰值。提示皮质冷冻性脑损伤后，损伤区周围星形胶质细胞GMFB表达增加。较少

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