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Treatment of rat C6 meningeal dissemination model by intrathecal injection o frecomvinant soluble FasL

鞘内注射常温可溶性FasL治疗大鼠C6脑膜播散模型

基本信息

批准号：
14571318
负责人：
SHIRAISHI Tetsuya
金额：
$ 2.5万
依托单位：
Saga University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

Fas (CD95) ligand (FasL) has the ability to induce apoptosis in Fas-expressing glioma cells by binding to with Fas: Several molecular species have been designed to be soluble Fas ligands for therapeutic purposes. Most of them possessing just only extracellular domain of Fas ligand (soluble FasL) cannot be used practically due to either insufficient biological activity or reduced productivity. We. successfully constructed a chimeric soluble. FasL by fusing a isoleucine zipper motif for se If-oligomerization and a FLAG sequence to the extracellular domain of the human Fas ligand (FIZ-shFasL). The cytotoxic effect of FIZ-shFasL on Jurkat cells was equivalent to that of membrane-bound FasL and approximately ten-fold stronger than that of agonistic anti-Fas antibody (CH-11). Expression of Fas and Bcl-2 on cell lines was determined using flow cytometry and compared with sensitivity to FIZ-shFasL. Flowcytometric analysis demonstrated that the differential Fas expression of human brain tumor cell lines partially correlated with levels of apoptosis through FIZ-shFasL. The intravenous administration of 1.0mg/kg of FIZ -shFasL resulted in a lethal effect in rats. A rat meningeal dissemination model with rat Fas-expressing C6 glioma cells can be treated successfully by intrathecal FIZ-shFasL administration without any apparent adverse effects. These results suggest that intrathecal administration of FIZ-shFasL maybe applied to the treatment of patients with leptomeningeal gliomatosis in the near future.

Fas（CD 95）配体（FasL）具有通过与Fas结合来诱导表达Fas的胶质瘤细胞凋亡的能力：已经设计了几种分子种类作为用于治疗目的的可溶性Fas配体。它们中的大多数仅具有Fas配体的胞外结构域（可溶性FasL），由于生物活性不足或生产率降低而不能实际使用。We.成功构建了嵌合可溶性。通过将用于自身寡聚化的异亮氨酸拉链基序和FLAG序列融合到人Fas配体的细胞外结构域（FIZ-shFasL）来表达FasL。FIZ-shFasL对Jurkat细胞的细胞毒作用与膜结合的FasL相当，并且比激动性抗Fas抗体（CH-11）强约10倍。采用流式细胞术测定细胞系上Fas和Bcl-2的表达，并与FIZ-shFasL的敏感性进行比较。流式细胞仪分析表明，Fas的差异表达的人脑肿瘤细胞系部分相关的凋亡水平通过FIZ-shFasL。静脉注射1.0mg/kg的FIZ-shFasL对大鼠有致死作用。用大鼠表达Fas的C6胶质瘤细胞的大鼠脑膜播散模型可以通过鞘内注射FIZ-shFasL成功地治疗，而没有任何明显的不良反应。这些结果表明，鞘内注射FIZ-shFasL可能在不久的将来应用于软脑膜胶质瘤病患者的治疗。