【作者】 花丽茹；

【导师】 宋铭忻；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 旋毛形线虫(Trichinella. Spiralis)简称旋毛虫,所致的旋毛虫病(Trichinosis)是呈世界性危害严重的人畜共患寄生虫病。旋毛虫排泄分泌(Excretory-Secretory antigen,ES)抗原,即旋毛虫生长过程中的排泄分泌产物,是旋毛虫检测和免疫的良好抗原,其保护作用是如今国内外众多学者研究旋毛虫免疫保护的热点之一。目前研究发现,某些寄生虫感染诱导的细胞凋亡(apoptosis)在疾病的发生机制中起重要作用,细胞凋亡在寄生虫发育过程中是必须的,它与寄生虫的生长、发育、感染和转归等密切相关。国内外对于旋毛虫病与细胞凋亡方面的研究甚少,本课题为国内首次以体外培养的小鼠胸腺淋巴细胞为实验对象,加入旋毛虫ES抗原刺激物,从生态学角度和生化特征等方面,通过对鼠胸腺淋巴细胞活性、细胞凋亡的形态学、生化特征及细胞周期等一系列指标的检测,进而证明旋毛虫肌幼虫ES抗原能够诱导鼠胸腺淋巴细胞产生凋亡。试验结果表明:1.通过台盼蓝排斥实验和MTT比色法测定了旋毛虫肌幼虫ES抗原对鼠胸腺淋巴细胞的半数抑制浓度(IC50),即:6h的IC50为24.96±0.46μg/ml、12h的IC50为17.36±0.68μg/ml、24h的IC50为11.09±0.17μg/ml、48h的IC50为5.35±0.86μg/ml。由此表明旋毛虫肌幼虫ES抗原对鼠类免疫细胞有损伤的作用,并且损伤作用随着时间的延长而增大,呈现出明显的时间效应。2.从细胞凋亡的形态学角度,利用AO/EB双荧光染色的方法证明旋毛虫肌幼虫ES抗原能够诱导鼠胸腺淋巴细胞发生凋亡。3.通过对细胞基因组DNA进行琼脂糖凝胶电泳并观察到明显的“梯形带”,从而进一步从生化特征角度定性地证明了旋毛虫肌幼虫ES抗原能够诱导鼠胸腺淋巴细胞发生凋亡。4.利用流式细胞术检测旋毛虫肌幼虫ES抗原对体外培养的鼠胸腺淋巴细胞DNA含量以及细胞周期的影响,说明旋毛虫肌幼虫ES抗原能够诱导鼠胸腺淋巴细胞发生凋亡。本试验从形态学和生化特征等角度入手,分析了旋毛虫肌幼虫ES抗原致鼠胸腺淋巴细胞凋亡的情况,研究发现旋毛虫ES抗原能够诱导鼠胸腺淋巴细胞发生凋亡,以上各方法均证明这种促凋亡作用呈现明显的时间效应,并由此说明旋毛虫ES抗原可能使宿主发生免疫抑制。这些研究结果对今后探索旋毛虫病的发病机制和提供防治对策具有重要意义。更多还原

【Abstract】 Trichinosis is a kind of serious zoonosis in the world which is caused by Trichinella spiralis. T. spiralis muscle larvae Excretory-Secretory antigen produced during the growth stage is a good antigen for detection and immunity of Trichinosis. The recent study indicated that apoptosis induced by parasitic infection plays an important role in mechanism of disease. Apoptosis is needed in parasites and related with growth, development, infection and turnover of parasites. There is rare information of Trichinosis and apoptosis internal and external. In order to study the relationship between apoptosis and infection of T. spiralis, mice thymus lymphocytes cultured in vitro was used as experiment materials in this study induced by T. spiralis muscle larvae ES antigen with some items, such as the activity of cells, cell cycle and morphology and biochemistry of apoptosis, detected. The results showed as follows:1. The IC50 of ES antigen on mice thymus lymphocytes was detected by trypan blue method and MTT method respectively. That is, 24.96±0.46μg/ml of 6h, 17.36±0.68μg/ml of 12h, 11.09±0.17μg/ml of 24h, 5.35±0.86μg/ml of 48h. The results showed that ES antigen is harmful to mice thymus lymphocytes, and the sensitivity increases along with time going.2. AO/EB double fluorescence coloration was used to detect apoptosis induced by T. spiralis muscle larvae ES antigen in mice thymus lymphocytes from the morphological aspect. It revealed that ES antigen induced lymphocytes death mainly by the way of apoptosis.3. The effectof ES antigen on DNA damage in mice thymus lymphocytes was detected by the method of DNA Ladder analysis. The result exhibited obvious ladder-shaped straps and showed that ES antigen caused apoptosis of lymphocytes by DNA damage, which exhibited time-dependent effect.4. Flow cytometry assay was used to detect cell cycle and DNA contents of mice thymus lymphocytes, and the result indicated that ES antigen induced apoptosis of mice thymus lymphocytes.The phenomenon of T. spiralis muscle larvae ES antigen induced mice thymus lymphocytes in vitro was detected in this study from morphology and biochemistry, and the results showed that ES antigen induced apoptosis of mice thymus lymphocytes with time-dependent effect, thereby, indicated ES antigen can cause immune suppression of host. This finding will play an important roll in pathogenesis, prevention and therapy of infection of T. spiralis afterward.更多还原