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

我们试图建立人T淋巴细胞趋化病毒I型(HTLV-I)前病毒DNA和转录本的同步定量检测系统，以检测HAM/TSP患者脑脊液细胞中的HTLV-I相关性脊髓病/热带痉挛麻痹(HAM/TSP)。建立了HTLV-I前病毒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外，我们还开发了一种新的技术来可视化HTLV-I前病毒DNA的单拷贝。用精制的高灵敏度探针进行荧光原位杂交(FISH)，成功地检测到HTLV-I前病毒DNA。FISH允许我们根据中期分裂和间期核内染色体上的信号数量来计算前病毒DNA的拷贝数。我们使用激光扫描细胞仪(LSC)来定量测量单个细胞中前病毒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前病毒DNA。LSC需要更高的灵敏度才能检测到较低的荧光强度。我们考察了同时检测前病毒DNA和mRNA或病毒蛋白的条件。最佳固定条件各不相同，但我们找到了适合同时检测的条件。用激光共聚焦扫描仪检测双标样品中的两种不同的荧光。LSC对相同强度的不同荧光有较好的检测能力，但不具备定量检测不同荧光强度的能力。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，并尝试测试已有的一些新技术。这些技术也被应用于包括HAM/TSP或HTLV-I相关疾病的不同疾病的其他样本的研究。

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.