GFAP GENE EXPRESSION IN HUMAN GLIOMA CELL

人胶质瘤细胞中的 GFAP 基因表达

• 批准号: 03670675
• 负责人: WASHIYAMA Kazuo
• 金额: $ 1.28万
• 依托单位: NIIGATA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03670675/
• 关键词: GFAP; Gene expression; Glioma; In situ hybridization; Northern blot; Cell line; gene expression; glioma; in situ hybridization; cell line

We have analyzed GFAP expression through successive passages of GFAP-positive human glioma cell line by using the immunohistochemistry and Northern blot hybridization. In one cell line (Case 1), the cultured cells showed stable expression of GFAP protein and mRNA from early to later passage levels. In the other cell line (Case 2), GFAP expression was shown to be less stable. At early passage, nearly 100% of the cultured cells displayed intense GFAP immunoreactivity. After passage 30, however, weakly to negatively stained cells increased gradually in number, and by passage 100, almost all cells had become negative for GFAP. A similar change in GFAP immunoreactivity during successive passages was also observed in each of 7 GFAP-positive clones isolated from this cell line at passage 30 and 50, suggesting that each cell had a tendency to reduce and finally stop its GFAP expression. Northern blot studies revealed that GFAP mRNA also change concomitantly in the parental and clonal cell lines, suggesting a regulatory change at the transcriptional level.GFAP mRNA have been analyzed in materials of one astrocytoma case, and of one anaplastic glioma case using in situ hybridization technique. Anti-sense oligomer specific for GFAP cDNA was selected from 3' non-coding region. The use of this probe with low stringency showed 3.4-kb band in human glioma cell lines by Northern blot hybridization. The positive grains specific for GFAP mRNA were distributed in Bergmann's glia in normal cerebellum, and in astrocytoma tissues, but not in anaplastic glioma tissues.

本研究应用免疫组化和北方印迹杂交技术，对胶质纤维酸性蛋白（GFAP）阳性的人脑胶质瘤细胞系进行了连续传代研究。在一个细胞系（病例1）中，培养的细胞显示从早期到后期传代水平GFAP蛋白和mRNA的稳定表达。在另一种细胞系中（病例2），GFAP表达显示不太稳定。在早期传代，近100%的培养细胞显示出强烈的GFAP免疫反应。然而，在第30代之后，弱染色至阴性染色的细胞数量逐渐增加，并且到第100代，几乎所有细胞都变成GFAP阴性。在连续传代过程中，在第30代和第50代从该细胞系分离的7个GFAP阳性克隆中也观察到GFAP免疫反应性的类似变化，这表明每个细胞都有减少并最终停止其GFAP表达的趋势。北方杂交结果显示，GFAP mRNA在亲本细胞系和克隆细胞系中也发生变化，提示在转录水平上的调节变化。从3'端非编码区选择特异性的反义寡聚体。用该探针进行北方印迹杂交，在人脑胶质瘤细胞系中显示3.4kb的条带。GFAP mRNA阳性颗粒在正常小脑Bergmann胶质细胞和星形细胞瘤组织中均有分布，而在间变性胶质瘤组织中无分布。