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

1.采用改良的原位末端标记法检测培养的脑胶质瘤细胞和脑肿瘤活检标本中核小体DNA片段化，并分析细胞凋亡相关基因产物的表达。TUNEL染色可见碎裂和浓缩的细胞核，染色的细胞核百分率(细胞凋亡指数)为0%~8.9%。结论1.Fas、Bcl2家族(Bcl2、Bclx、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(T98G、U251和A172)中的作用。免疫组织化学和流式细胞术分析表明，这三种细胞株都高度表达Fas，但只有T98G细胞在Fas单抗(50 ng/ml)作用下才出现核碎裂、DNA凝胶电泳梯形条带和存活细胞减少等凋亡变化。我们一直在使用OK-432激活的外周血单核细胞(OK-MC)对恶性脑肿瘤患者应用过继免疫治疗方案。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T98G胶质瘤细胞的杀伤活性。ZB4单抗可明显抑制OK-MCs诱导的T98G细胞的凋亡，ZB4单抗可与Fas结合并阻断Fas配体与Fas的结合。这些结果表明，OK-MC的细胞毒作用可能部分是由Fas配体/Fas系统介导的。

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.