Abstract：
Microsporidia are obligate intracellular pathogens that can infect many vertebrate and invertebrate hosts. While the Microsporidia phylum was defined as protozoa until the 1990s, it has been associated with fungi in line with the data obtained as a result of phylogenetic and molecular analyzes in recent years. Although approximately 200 genera and 1400 Microsporidia species related to these genera have been reported to date, only 14 species are known to cause infection in humans. Encephalitozoon intestinalis is one of the most frequently detected species in humans and causes serious clinical conditions in immunosuppressed individuals. Little information is available about the immunology of this infection. This study was aimed to investigate the changes in Toll-Like receptor (TLR) gene expressions in Madin-Darby canine kidney (MDCK) cells treated with E.intestinalis spores. Three groups were formed in the study. In the first group, only the medium prepared for E.intestinalis was added to the MDCK cells. In the second group, 108 live spores waiting at +4 °C were added. In the third group, 108 heat-inactivated spores were added. All three groups were incubated at 37ºC with 5% CO2 . RNA isolation and cDNA synthesis were performed from samples taken from these groups at the 1st, 3rd, 6th, 12th and 24th hours. Expression of TLR1-10 genes from the obtained cDNAs was evaluated by real-time polymerase chain reaction (Rt-PCR). GAPDH and ACTB genes were used as housekeeping genes in the study. Target genes were normalized by taking the average of these two genes and statistical analysis was performed by applying the 2-ΔΔCt formula. Genes detected above the threshold value (threshold 1) were considered to have increased expression. Genes detected below the threshold value were considered to have decreased expression. The growth of the live and inactive spores were followed simultaneously with the experimental groups. Approximately two weeks after the start of the culture, it was observed that E.intestinalis grew in the culture with live spore, but did not grow in the culture with inactivated spores. No statistically significant change was observed in gene expressions in the inactivated spore group. In the live spore group, a significant increase was seen in the expression of only two genes. These genes were TLR3 and TLR4. It was observed that there was a significant increase in TLR3 gene expression at the first hour (1.6-fold of control group) but the expression level started to decrease at the third hour (1.4-fold of control group) and returned to the control level at the sixth hour. It was observed that TLR4 gene expression continued parallel to the control until the 24th hour and increased significantly (2.1-fold of control group) at the 24th hour. In conclusion, this study is the f irst report in which the changes in ten different TLR gene expressions were evaluated at different times in MDCK cells stimulated with E.intestinalis and the change in TLR3 gene expression.
摘要：
微孢子虫是一种专性细胞内病原体，可以感染许多脊椎动物和无脊椎动物宿主。直到1990年代，微孢子虫门才被定义为原生动物，与近年来通过系统发育和分子分析获得的数据一致，它与真菌有关。尽管迄今为止已经报道了大约200属和1400个与这些属有关的微孢子虫物种，已知只有14种物种会导致人类感染。小肠动物是人类中最常检测到的物种之一，并在免疫抑制的个体中引起严重的临床状况。关于这种感染的免疫学的信息很少。本研究旨在研究肠球菌孢子处理的Madin-Darby犬肾（MDCK）细胞中Toll样受体（TLR）基因表达的变化。在研究中形成三组。在第一组中,仅将为肠球菌制备的培养基添加到MDCK细胞中。在第二组中,添加108个在+4°C下等待的活孢子。在第三组中,添加108个热灭活的孢子。所有三组均在37ºC和5%CO2下孵育。RNA分离和cDNA合成从这些组的样品在1,3rd,6th,12小时和24小时。通过实时聚合酶链反应(Rt-PCR)评价来自获得的cDNA的TLR1-10基因的表达。GAPDH和ACTB基因用作研究中的管家基因。通过取这两个基因的平均值将靶基因标准化,并通过应用2-ΔΔCt公式进行统计分析。检测到的高于阈值(阈值1)的基因被认为具有增加的表达。检测到低于阈值的基因被认为具有降低的表达。与实验组同时跟踪活孢子和非活性孢子的生长。培养开始大约两周后，观察到肠球菌在具有活孢子的培养物中生长，但在具有失活孢子的培养物中没有生长。在灭活孢子组中，基因表达没有观察到统计学上的显着变化。在活孢子组中，只有两个基因的表达显着增加。这些基因分别为TLR3和TLR4。观察到TLR3基因表达在第一小时显著增加(对照组的1.6倍),但表达水平在第三小时开始降低(对照组的1.4倍),并在第六小时恢复到对照水平。观察到TLR4基因表达与对照平行持续到第24小时,并且在第24小时显著增加(对照组的2.1倍)。总之,这项研究是首次报道，其中在不同时间评估了肠球菌刺激的MDCK细胞中10种不同TLR基因表达的变化以及TLR3基因表达的变化。
