GENE EXPRESSION RESPONDED TO FAS STIMULATION IN TRAYPANOSOMA CRUZI INFECTED HOST CELLS USING DNA MICROARRAY TECHNIQUE

使用 DNA 微阵列技术在 TRAYPANOSOMA CRUZI 感染的宿主细胞中响应 FAS 刺激的基因表达

• 批准号: 14570221
• 负责人: SHIMADA Junko
• 金额: $ 2.62万
• 依托单位: JUNTENDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570221/
• 关键词: DNA MICROARRAY; TRYPANOSOMA; APOPTOSIS INHIBITION; FAS; C-FLIP; INFECTED ANIMAL MODEL; cFLIP

Thypanosoma cruzi the protozoan parasite that causes Chagas' disease in Latin America. We reported that T.cruzi infection inhibits Fas-mediated apoptosis in host cells, and this inhibition is thought to be a defense strategy for the parasite to escape from host immune responses. To elucidate a molecular mechanism of the inhibition, we determined the time courses of transcriptional changes in uninfected (control) and T.cruzi infected cells using DNA microarray technology. Human fibrosarcoma cells were infected with T.cruzi, cultured for 3-4 days and stimulated by anti-Fas antibody. Total RNA was extracted and converted to first-strand cDNA: cDNA was labeled with Cy5-dUTP (control) or Cy3-dUTP (infected) and hybridized to human apoptosis chips consisting of 164 apoptosis related genes. Apoptosis inhibitory genes (apoptosis inhibitor 1 and c-FLLIP) and caspases (caspase-1, -4, and -7) showed higher expression levels in T.cruzi infected than in control cells at 10 to 30 mm after Fas stimulation. Further, the fluorescence intensities of I-kB, bcl-W and cell cycle related genes were higher in infected cells at 2 to 24 h after the induction. To determine the consequences of a T.cruzi infection on apoptosis in vivo, we infected Wistar rats with either the T.cruzi Sylvio-X10/7 clone or the Sylvio-X10/4 clone to simulate an acute (lethal) or chronic disease model, respectively. At predetermined times post-infection, paraffin sections of the rats' hearts were processed using the TUNEL reaction to detect apoptotic cells. In the lethal disease model, inflammatory lymphocytes were found around T.cruzi-infected muscle cells at 7 days post-infection. Surprisingly, however, although some of the lymphocytes were TUNEL positive, T.cruzri-infected muscle cells were TUNEL negative. Furthermore, in the chronic disease model it was difficult to find either T.cruzi infected cells or apoptotic cells in the heart sections.

克氏锥虫：在拉丁美洲引起恰加斯病的原生动物寄生虫。我们报道了克氏锥虫感染抑制fas介导的宿主细胞凋亡，这种抑制被认为是寄生虫逃避宿主免疫应答的一种防御策略。为了阐明抑制的分子机制，我们使用DNA微阵列技术测定了未感染（对照）和克氏锥虫感染细胞的转录变化的时间过程。用克氏锥虫感染人纤维肉瘤细胞，培养3-4天，用抗fas抗体刺激。提取总RNA转化为第一链cDNA，用Cy5-dUTP（对照）或Cy3-dUTP（感染）标记cDNA，杂交到包含164个凋亡相关基因的人凋亡芯片上。凋亡抑制基因（凋亡抑制剂1和c- flip）和caspase （caspase-1、-4和-7）在Fas刺激后10 ~ 30mm感染T.cruzi细胞中的表达水平高于对照细胞。诱导后2 ~ 24 h，感染细胞中I-kB、bcl-W和细胞周期相关基因的荧光强度较高。为了确定克氏锥虫感染对体内细胞凋亡的影响，我们分别用克氏锥虫Sylvio-X10/7克隆或Sylvio-X10/4克隆感染Wistar大鼠，模拟急性（致死）或慢性疾病模型。在感染后的预定时间，使用TUNEL反应处理大鼠心脏石蜡切片以检测凋亡细胞。在致死性疾病模型中，感染后7天在克氏锥虫感染的肌肉细胞周围发现炎性淋巴细胞。然而，令人惊讶的是，尽管一些淋巴细胞TUNEL阳性，但感染t .cruzri的肌肉细胞TUNEL阴性。此外，在慢性疾病模型中，在心脏切片中很难发现克氏杆菌感染细胞或凋亡细胞。

项目成果

嶋田淳子: "Trypanosoma cruziと宿主細胞のアポトーシスをめぐるせめぎあい"The 72^<nd> Annual Meeting of The Japanese Society of Parasitology. 73 (2002)

Junko Shimada：“克氏锥虫与宿主细胞凋亡之间的斗争”第 72 届日本寄生虫学会年会 73（2002 年）。

Shimada J.: "Pathology and apoptosis: parasite infection."Rinsho Men-eki. 38. 395-403 (2002)

Shimada J.：“病理学和细胞凋亡：寄生虫感染。”Rinsho Men-eki。

Uchiyama N.: "Trypanocidal terpenoids from Laurus nobilis L."Chem.Pharm.Bull.. 50. 1514-1516 (2002)

Uchiyama N.：“月桂树中的杀锥虫萜类化合物”Chem.Pharm.Bull.. 50. 1514-1516 (2002)

嶋田淳子, 北潔: "病態とアポトーシス:寄生虫感染"臨床免疫. 38巻増刊. 395-403 (2002)

Junko Shimada，Kiyoshi Kita：“病理学和细胞凋亡：寄生虫感染”临床免疫学第 38 卷特别版（2002 年）。

SHimada J.: "The struggle between the host cell and Typanosoma cuzi; Infected host cell may have promoted own apoptosis but the parasite inhibits it."The 72^<nd> Annual Meeting of The Japanese Society of Parasitology. 73 (2002)

Shimada J.：“宿主细胞和Typanosoma cuzi之间的斗争；受感染的宿主细胞可能促进自身细胞凋亡，但寄生虫抑制它。”日本寄生虫学会第72届年会。