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

克氏锥虫是一种原生动物寄生虫，在拉丁美洲引起查加斯病。我们报道了克氏毛滴虫感染抑制宿主细胞中Fas介导的细胞凋亡，这种抑制被认为是寄生虫逃避宿主免疫反应的一种防御策略。为了阐明这种抑制的分子机制，我们利用DNA芯片技术检测了未感染(对照)和感染的克氏毛滴虫细胞转录变化的时间进程。用克氏毛滴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，与含有164个凋亡相关基因的人凋亡芯片杂交。在Fas刺激后10~30 mm处，凋亡抑制基因(凋亡抑制因子1和c-FLLIP)和caspase(caspase-1、-4和-7)在感染的克氏毛滴虫细胞中的表达水平高于对照细胞。此外，在诱导后2~24小时，感染细胞的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，我们用克氏锥虫Sylvio-X10/7克隆或Sylvio-X10/4克隆感染Wistar大鼠，分别模拟急性(致命)或慢性疾病模型。在感染后的预定时间，用TUNEL反应处理大鼠心脏的石蜡切片，以检测凋亡细胞。在致死性疾病模型中，感染后7天，克氏毛滴虫感染的肌肉细胞周围可见炎性淋巴细胞。然而，令人惊讶的是，尽管一些淋巴细胞TUNEL阳性，但感染克鲁兹里毛滴虫的肌肉细胞TUNEL阴性。此外，在慢性病模型中，很难在心脏切片中发现克氏毛滴虫感染细胞或凋亡细胞。

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.