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Glial Response and Neuronal Cell Death Induced by HTLV-I Infected Cells

HTLV-I 感染细胞诱导的神经胶质反应和神经元细胞死亡

基本信息

批准号：
07670717
负责人：
SHIRABE Susumu
金额：
$ 1.54万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
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

项目摘要

Although pathogenesis of HTLV-I associated myelopathy has been extensively studied, the mechanism of cell damage constructing central nervous system is still unclear. On the other hand, primary culture procedures of neuron and glial cells are now established and available for a cellular model for viral infections.In this study, we tried to investigate the mechanisms of in-vitro neuronal and glial cell death as a result of HTLV-I infection.First of all, we investigated the effect of HTLV-I infected cells such as MT-1, MT-2, HCT-1, W7TM-I and lymphocytes derived from HAM patients against the primary cerebellar granular cells of rats. 1. Supernatants of MT-1 and MT-2 damaged the cerebellar granular cells in dose-dependent manner. The evaluation was performed by MTT assay and fluorescin diacetate assay. Anti-P40tax (tax protein) antibody failed to neutralized this cell damage, suggesting p40tax may affecting expression of cellular factors such as inflammatory cytokines.Tax gene which is integrated into expression vector was transfected into primary astrocytes, microglia and cerebellar granular cells, respectively. Only few cells can be transfected by lipofectoamine system in each experiment, indicating this method is not available for primary cells.So, we proceeded to use cell lines to try to determine whether tax-transfected cells can produce cytotoxic factors. Macrophage cell line, U937 supposed to be a model of microglial cells were transfected with tax gene. We disclosed that tax-transfected U937 were over-expressing inducible nitric oxide synthase (iNOS) by semi-quantitative PCR.Direct evidence for over-production of NO was measured by detecting using chemiluminescence and adding Vanadium (III)(FES-450, Scholar-Tee Co., Ltd., Japan). These exaggerated NO production were enhanced by gamma-IFN.This result indicating that microglia/macrophage might damage neuronal cells by exaggerated NO production.

虽然HTLV-I相关性脊髓病的发病机制已被广泛研究，但构成中枢神经系统的细胞损伤机制仍不清楚。另一方面，神经元和神经胶质细胞的原代培养方法现已建立，并可用于病毒感染的细胞模型。在本研究中，我们试图研究HTLV-I感染导致的体外神经元和神经胶质细胞死亡的机制。首先，我们研究了HTLV-I感染的细胞如MT-1，MT-2，HCT-1，W7 TM-I和HAM患者来源的淋巴细胞对大鼠小脑原代颗粒细胞的作用。1. MT-1和MT-2培养上清对小脑颗粒细胞的损伤呈剂量依赖性。采用MTT比色法和荧光素二乙酸酯比色法进行评价。抗P40 tax（tax蛋白）抗体未能中和这种细胞损伤，提示p40 tax可能影响炎症因子等细胞因子的表达。将整合于表达载体中的Tax基因分别转染原代星形胶质细胞、小胶质细胞和小脑颗粒细胞。由于脂质体介导的细胞转染率较低，说明该方法不适用于原代细胞，因此，我们尝试用细胞系来检测转染tax的细胞是否能产生细胞毒因子。以巨噬细胞系U937为小胶质细胞模型，转染tax基因。我们通过半定量PCR公开了tax转染的U937过表达诱导型一氧化氮合酶（iNOS）。通过使用化学发光检测并加入VanCl 4（III）（FES-450，Scholar-Tee Co.，有限公司、日本）。γ-IFN可增强这些NO的过度产生，提示小胶质细胞/巨噬细胞可能通过过度产生NO损伤神经细胞。