Development of simultaneous and quantitative detection system of human T-lymphotropic virus type I proviral DNA and transcripts in cerebrospinal fluid cells from patients with human T-lymphotropic virus type I-associated myelopathy/tropical spastic parapa

人类嗜T淋巴细胞病毒I型相关性脊髓病/热带痉挛性帕拉帕患者脑脊液细胞中人类嗜T淋巴细胞病毒I型前病毒DNA和转录物同步定量检测系统的开发

• 批准号: 12670613
• 负责人: MORITOYO Takashi
• 金额: $ 2.18万
• 依托单位: Kagoshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670613/
• 关键词: HTLV-I; HAM; cerebrospinal fluid cells; in situ hybridization; immunohistochemistry; Laser Scanning Cytometer; HTLV-I

We tried to develop the simultaneous and quantitative detection system of human T-lymphotropic virus type I (HTLV-I) proviral DNA and transcripts in cerebrospinal fluid cells from patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The fluorescence detection system of HTLV-I proviral DNA, mRNA and viral proteins were established. In addition to the highly sensitive detection system of HTLV-I mRNA and viral proteins that we developed, a novel technique to visualize a single copy of HTLV-I proviral DNA was developed. HTLV-I proviral DNA was successfully detected by fluorescence in situ hybridization (FISH) using refined highly sensitive probes. FISH allowed us to count the number of copies of proviral DNA from the number of signals on chromosomes within a metaphase spread and the interphase nucleus. We used Laser Scanning Cytometer (LSC) to measure the total amount of fluorescence of proviral DNA, mRNA or viral proteins from an individual cell quantitatively. But LSC did not have enough sensitivity to detect the proviral DNA. LSC was needed to be more sensitive to detect the lower intensity of fluorescence. We examined the condition of simultaneous detection of proviral DNA and mRNA or viral proteins. The best fixative condition was different from each other, but we found the appropriate condition to detect simultaneously. LSC was used to detect the two different fluorescence from double-labeled samples. LSC was good to detect the same intensity of different fluorescence, but did not have enough ability to detect the different amount of fluorescence quantitatively. We started to collect and stock the samples of cerebrospinal fluid cells from patients with HAM/TSP and HTLV-I-associated diseases, and tried to test the new some techniques that had been available. These techniques were also applied to investigate the other samples including different diseases from HAM/TSP or HTLV-I-associated diseases.

我们试图开发 HTLV-I 相关脊髓病/热带痉挛性截瘫 (HAM/TSP) 患者脑脊液细胞中人类 T 淋巴细胞病毒 I 型 (HTLV-I) 前病毒 DNA 和转录物的同步定量检测系统。建立了HTLV-I原病毒DNA、mRNA和病毒蛋白的荧光检测体系。除了我们开发的 HTLV-I mRNA 和病毒蛋白的高灵敏度检测系统之外，还开发了一种可视化 HTLV-I 前病毒 DNA 单拷贝的新技术。使用精制的高灵敏度探针通过荧光原位杂交 (FISH) 成功检测到 HTLV-I 前病毒 DNA。 FISH 使我们能够根据中期扩散和间期细胞核内染色体上的信号数量来计算原病毒 DNA 的拷贝数。我们使用激光扫描细胞仪 (LSC) 定量测量单个细胞中原病毒 DNA、mRNA 或病毒蛋白的荧光总量。但 LSC 没有足够的灵敏度来检测原病毒 DNA。 LSC 需要更灵敏地检测较低强度的荧光。我们检查了同时检测原病毒DNA和mRNA或病毒蛋白的情况。最佳固定条件各不相同，但我们找到了同时检测的合适条件。 LSC 用于检测双标记样品的两种不同荧光。 LSC能够很好地检测相同强度的不同荧光，但没有足够的能力来定量检测不同量的荧光。我们开始收集和储存 HAM/TSP 和 HTLV-I 相关疾病患者的脑脊液细胞样本，并尝试测试现有的一些新技术。这些技术还应用于研究其他样本，包括 HAM/TSP 或 HTLV-I 相关疾病的不同疾病。

项目成果

Sugimoto C: "nef gene is required for robust productive infection by simian immunodeficiency virus of T-cell-rich paracortex in lymph nodes."J Virol. 77. 4169-4180 (2003)

Sugimoto C：“nef 基因是猿免疫缺陷病毒对淋巴结中富含 T 细胞的副皮质进行强力有效感染所必需的。”J Virol。

Toshio Matsuzaki: "HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) with amyotrophic lateral sclerosis-like manifestations."J.Neurovirol. 6. 544-548 (2000)

Toshio Matsuzaki：“HTLV-I 相关脊髓病 (HAM)/热带痉挛性截瘫 (TSP) 伴有肌萎缩侧索硬化症样表现。”J.Neurovirol。

Yoshida Y: "Intraventricular administration of the neurotrophic factor midkine ameliorates hippocampal delayed neuronal death following transient forebrain ischemia in gerbils."Brain Res. 894. 46-55 (2001)

Yoshida Y：“神经营养因子中期因子的脑室内给药可改善沙鼠短暂前脑缺血后海马延迟性神经元死亡。”Brain Res。

Moe Moe Aye: "Histopathological analysis of four autopsy cases of HTLV-I-associated myelopathy/tropical spastic paraparesis : inflammatory changes occur simultaneously in the entire central nervous system."Acta Neuropathol.Berl.. 100. 245-252 (2000)

Moe Moe Aye：“HTLV-I 相关脊髓病/热带痉挛性截瘫四例尸检病例的组织病理学分析：整个中枢神经系统同时发生炎症变化。”Acta Neuropathol.Berl.. 100. 245-252 (2000)

Intraventricular administration of the neurotrophic factor midkine ameliorates hippocampal delayed neuronal death following transient forebrain ischemia in gerbils

脑室内注射神经营养因子中期因子可改善沙鼠短暂前脑缺血后海马迟发性神经元死亡

• DOI: 10.1016/s0006-8993(00)03209-1
• 发表时间: 2001-03-09
• 期刊: Brain Research
• 影响因子: 2.9
• 作者: Y. Yoshida;S. Ikematsu;T. Moritoyo;M. Goto;J. Tsutsui;S. Sakuma;M. Osame;T. Muramatsu
• 通讯作者: T. Muramatsu