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

1、人神经胶质成熟因子- γ基因启动子活性分析我们确定了9.5 kb hGMFG基因的组织结构，并对其启动子活性进行了表征。在起始密码子上游的226 bp序列中，第一个外显子的5'侧区没有TATA或CAAT盒子。引物延伸分析和5‘ race （cDNA 5’端快速扩增）在Kozak共识序列中从第一个ATG密码子的-84到-70个核苷酸区域内鉴定出多个转录起始位点。通过双荧光素酶报告系统将一系列缺失构建体转染到大鼠星形胶质细胞来源的ACT-57细胞中，确定了核心启动子区域。2、建立神经胶质成熟因子β (GMFB)和γ (GMFG)的双位点酶免疫分析法（EIA）利用兔培养的特异性抗体，建立了神经胶质成熟因子β (GMFB)和γ (GMFG)的双位点酶免疫分析法（EIA）。这些检测系统使我们能够在人和大鼠样本中识别GMFB和GMFG (GMFs)，并用于研究人和大鼠GMFs的组织分布和血清浓度。在大鼠中，除了脊髓外，在中枢神经系统以及胸腺和结肠中发现了相对高水平的GMFB。在胸腺、脾脏和结肠中发现了较高水平的GMFG。转基因食品在人体内的分布与在大鼠体内的分布相似。大鼠血清GMFG浓度在4周龄时达到最大值。在出生后的前30天内，雄性和雌性的甲氧基甲氧基含量急剧下降。另一方面，血清GMFB浓度随年龄变化不显著。同样，人血清中GMFG浓度在21-30岁组最高，在30岁组开始迅速下降。相比之下，GMFB浓度在此期间没有明显变化。在人类和大鼠血清中转基因食品的水平没有显著的性别差异。3、低温脑损伤后神经胶质成熟因子β的表达分析采用免疫组织化学、逆转录聚合酶链反应（RT-PCR）、Western blotting和酶免疫分析法研究低温脑损伤后56 d内神经胶质成熟因子β的表达。免疫组织化学分析显示，损伤周围gfap阳性的星形胶质细胞表达GMFB蛋白，在损伤后14天达到峰值。假手术动物脑中GMFB免疫反应性弱星形细胞表达。低温损伤（CI）在7天后诱导损伤侧GMFB mRNA表达，在14天后达到最大值。Western blot显示GMFB蛋白的诱导从损伤后1天开始，一直持续到损伤后14天。在酶免疫分析中，损伤侧脑组织提取物GMFB蛋白浓度在损伤后14天达到峰值，而血清GMFB蛋白浓度在损伤后1天达到峰值。这些数据表明，皮层低温脑损伤后，损伤区周围星形胶质细胞中GMFB的表达增加。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