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

1、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天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。2、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 -70个核苷酸范围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。我们还确定了小鼠GMFG基因的组织结构，并克隆了第一外显子的5'-侧翼区域。荧光素酶法分析启动子活性。

1, 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.2, 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.We have also determined the organization of the mouse GMFG gene and cloned the 5'-flanking region of the first exon. The promoter activity was analyzed using luciferase assay.