Molecular biological studies on the characterization of novel human endogenous retrovirus associated with autoimmune diseases.

与自身免疫性疾病相关的新型人内源性逆转录病毒特征的分子生物学研究。

• 批准号: 12670419
• 负责人: INOUE Tetsufumi
• 金额: $ 2.43万
• 依托单位: Tokyo University of Foreign Studies
• 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-12670419/
• 关键词: Endogenous retrovirus; methylation; 自己免疫病; レトロウイルス

Human endogenous retroviruses (HERV) sequences are dispersed throughout the human genome, which may be useful as a genetic marker for human genome research. In addition, it is possible that HERV may contribute to the pathogenesis of autoimmune diereses, either by activating the surrounding genes via its promoter activiites or generating virus proteins and thereby yielding immune complexes. Since demethylation of CpG islands is Associated with transcriptional activities, demethylated HERV may be transcriptionally active in vivo. In the present study, we investigated the methylation pattern of genes surrounding HERV, using suppression PCR method, for the purpose of obtaining the information as to the HERV associated with autoimmune diseases. In brief, genomic DNA was digested with methylation-sensitive restriction enzyme, HpaII, and was ligated to adapter primer. PCR was subsequently performed using adapter and HERV-specific primer. As control, methylation-independent enzyme, Mspl, was used. The fragment was ananalyzed by an automated sequencer. When we compare the results between DNA isolated from neutrophils and that of lymph nodes, the pattern of MspI fragment was the same, indicating that there exists no genetic difference, in terms of HpaII/MspI RFLP, between the samples. In contrast, HpaII fragment Pattern was different from each other, presumably reflecting the epigenetic difference (methylation) between them. In RA, HpaII fragment pattern of peripheral blood was different from that of synovila fluid cells. There was also a difference of HpaII pattern of neutrophils, before and after anti-rheumatic treatments. Suppression PCR may thus be a convenient way, to study the difference of methylation pattern around HERV in various autoimmune diseases,

人类内源性逆转录病毒(HERV)序列广泛分布于人类基因组中，可作为人类基因组研究的遗传标记。此外，HERV可能通过其启动子激活周围基因或产生病毒蛋白从而产生免疫复合体，从而参与自身免疫性疾病的发病。由于CpG岛的去甲基化与转录活性有关，去甲基化的HERV在体内可能具有转录活性。在本研究中，我们用抑制性聚合酶链式反应的方法研究了HERV周围基因的甲基化模式，以获得与自身免疫性疾病相关的HERV的信息。简而言之，基因组DNA用甲基化敏感的限制性内切酶HpaII酶切，并连接到适配引物上。随后使用适配器和HERV特异性引物进行聚合酶链式反应。以甲基化非依赖性酶MSPL作为对照。用自动测序仪对该片段进行分析。从中性粒细胞和淋巴结中提取的DNA进行比较时，MspI片段的图谱是相同的，这表明两个样本之间在HpaII/MspI RFLP方面没有遗传差异。相反，HpaII片段模式彼此不同，可能反映了它们之间的表观遗传差异(甲基化)。类风湿性关节炎患者外周血和滑膜细胞的HpaII片段模式不同。抗风湿治疗前后中性粒细胞HpaII亚型也有差异。因此，抑制性聚合酶链式反应可能是一种方便的方法，可以研究各种自身免疫性疾病中HERV基因甲基化模式的差异。

项目成果

Sawada T, Hashimoto S, et al.: "Inhibition of L-leucine methyl ester mediated killing of THP-1, a human monocytic cell line, by a new anti-inflammatory drug, T614."Immunopharmacology. 49・3. 285-294 (2000)

Sawada T、Hashimoto S 等人：“新型抗炎药 T614 抑制 L-亮氨酸甲酯介导的 THP-1 杀伤作用”，免疫药理学 49・3。 294（2000）

Sawada T, Hashimoto S, et al.: "Inhibition of L-leucine methyl estwr mediated killing of THP-1, a human monocytic cell line, by a new anti-inflammatory drug, T614"Immunopharmacology. 49・3. 285-294 (2000)

Sawada T、Hashimoto S 等人：“新型抗炎药 T614 抑制 L-亮氨酸甲基 estwr 介导的 THP-1 杀伤”免疫药理学 49・3。 (2000)

SawadaT, Hashimoto S, Tohma S, Nishioka Y, Nagai T, Sato T, Ito K, Inoue T, Iwata M, Yamamoto K.: "Inhibition of L-leucine methyl ester mediated killing of THP-1, a humanmonocytic cell line, by a new anti-inflammatory drug, T614"Immuriopharmacology. 49(3)

SawadaT、Hashimoto S、Tohma S、Nishioka Y、Nagai T、Sato T、Ito K、Inoue T、Iwata M、Yamamoto K.：“抑制 L-亮氨酸甲酯介导的 THP-1（一种人类单核细胞系）的杀伤作用，

Sawada T,Hashimoto S, Inoue T et al: "Inhibition of L-leucine methyl ester mediated killing of THP-1, a human monocytic cell line, by a new drug T614."Immunopharmacology. 49(3). 285-294 (2000)

Sawada T、Hashimoto S、Inoue T 等人：“新药 T614 抑制 L-亮氨酸甲酯介导的人类单核细胞系 THP-1 的杀伤作用。”免疫药理学。