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

基本信息

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

项目摘要

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.

我们试图开发来自人类T-淋巴机病毒（HTLV-I）前动物（HTLV-I）前动物DNA的同时定量检测系统，并在患有HTLV-I相关性骨髓病/热带痉挛性刺激性的患者的脑脊髓流体细胞中的转录和转录本（HAM/TSP）。建立了HTLV-I病毒DNA，mRNA和病毒蛋白的荧光检测系统。除了我们开发的HTLV-I mRNA和病毒蛋白的高度敏感的检测系统外，还开发了一种可视化HTLV-I前病毒DNA副本的新技术。使用高度敏感的探针通过荧光原位杂交（FISH）成功检测了HTLV-1病毒DNA。 FISH使我们能够从中期扩散和相间核中的染色体信号数量计算前病毒DNA的副本数量。我们使用激光扫描细胞仪（LSC）来测量来自单个细胞的病毒DNA，mRNA或病毒蛋白的荧光总量。但是LSC没有足够的灵敏度来检测病毒DNA。 LSC需要更敏感以检测荧光的较低强度。我们检查了前病毒DNA和mRNA或病毒蛋白的同时检测条件。最佳的固定条件彼此不同，但是我们发现了同时检测的适当条件。 LSC用于检测双标签样品的两种不同的荧光。 LSC很好地检测了不同荧光的相同强度，但没有足够的能力来定量检测不同量的荧光。我们开始收集并存储来自HAM/TSP和HTLV-I相关疾病患者的脑脊液细胞样品，并试图测试新的一些可用技术。这些技术还用于研究其他样本，包括来自HAM/TSP或HTLV-I相关疾病的不同疾病。