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STUDY OF DNA FRAGMENTATION AND APOPTOSIS-RELATED GENE PRODUCTS IN HUMAN BRAIN TUMORS AND ESTABLISHMENT OF APOPTOSIS-INDUCING THERAPY.

人脑肿瘤中DNA片段和凋亡相关基因产物的研究及凋亡诱导疗法的建立。

基本信息

批准号：
07457319
负责人：
TABUCHI Kazuo
金额：
$ 4.35万
依托单位：
SAGA MEDICAL SCHOOL
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07457319/
关键词：
Brain tumor apoptsis TUNEL Fas Fas ligand ICE Bcl-2 OK-432 養子免疫療法 ICE inhibitor p53 p16 p21 Bcl-x

项目摘要

1. We detected the internucleosomal DNA fragmentation by modified TUNEL staining and analyzed the expression of apoptosis-related gene products in cultured glioma cells and biopsied brain tumor specimens. The fragmented and condensed nuclei were clearly stained for TUNEL and the percentages of stained nuclei (apoptotic index) were ranging from 0% to 8.9%. Fas, Bcl-2 families (Bcl-2, Bcl-x and Bax) and ICE families (ICE,Ich-1) were found to be involved in tumorigenesis of certain brain tumors.2. We investigated the role of the Fas ligand-Fas system in human glioblastoma cell lines (T98G,U251 and A172).Immunohistochemical and flow cytometrical analyzes revealed that all three cell lines expressed Fas highly, however, apoptotic changes such as nuclear fragmentation, the DNA ladder pattern on gel electrophoresis, and reduction of viable cells were observed only in T98G cells by treatment of Fas mAb (50ng/ml).3. We have been applying an adoptive immunotherapy protocol to patients with malignant brain tumors using OK-432-activated peripheral blood mononuclear cells (OK-MCs). To elucidate the mechanism of OK-MCs' cytotoxicity, we examined the cytocidal activity of OK-MCs against Fas expressing T98G glioma cells.Apoptosis of T98G cells induced by treatment with OK-MCs was unequivocally inhibited by the treatment of T98G cells with ZB4 monoclonal antibody, which binds to Fas and blocks the binding of Fas ligand to Fas. These results indicate that the cytotoxic activity of OK-MCs via apoptosis seems to be partly mediated by the Fas ligand/Fas system.

1.采用改良TUNEL法检测神经胶质瘤细胞核小体DNA片段化，并分析胶质瘤细胞凋亡相关基因产物的表达。断裂和凝聚的细胞核被TUNEL清楚地染色，染色细胞核的百分比（凋亡指数）范围为0%至8.9%。Fas、Bcl-2家族（Bcl-2、Bcl-x和Bax）和ICE家族（ICE、Ich-1）参与了某些脑肿瘤的发生.我们研究了Fas配体-Fas系统在人胶质母细胞瘤细胞系中的作用，免疫组化和流式细胞术分析显示，三种细胞系均高表达Fas，但均出现凋亡改变，如核碎裂、凝胶电泳DNA梯状条带、Fas单抗（50 ng/ml）处理T98 G细胞后，仅T98 G细胞活细胞数减少.我们一直在应用过继免疫治疗方案，恶性脑肿瘤患者使用OK-432激活的外周血单核细胞（OK-MCs）。为了阐明OK-MCs的细胞毒作用机制，我们检测了OK-MCs对表达Fas的T98 G胶质瘤细胞的杀伤活性，结果表明，与Fas结合并阻断Fas配体与Fas结合的ZB 4单克隆抗体能明显抑制OK-MCs诱导的T98 G细胞凋亡。这些结果表明，OK-MCs通过凋亡的细胞毒活性似乎部分由Fas配体/Fas系统介导。