辐照致弱捻转血矛线虫幼虫形态特征及基因表达谱分析

发布时间：2018-01-25 09:24

本文关键词： 捻转血矛线虫辐照致弱幼虫免疫原性细胞死亡凋亡　出处：《上海师范大学》2017年硕士论文　论文类型：学位论文

【摘要】：捻转血矛线虫属毛圆科血矛属线虫,是山羊等反刍动物的主要胃肠道寄生虫之一,患病动物通常表现为贫血、消瘦甚至死亡,造成巨大经济损失。目前的主要防治方法是使用化学药物驱虫,但随着抗药性虫株的的出现,化学药品治疗正受到严重的挑战。人们正在努力寻求新的防控策略,其中免疫预防用分子疫苗的研发尤其受到重视,但迄今没有一种高保护性的分子疫苗能够推广应用于现场。以前的研究发现,应用γ射线和紫外线照射致弱的捻转血矛线虫和蛇形毛圆线虫幼虫免疫家畜都获得了较高的保护性效果,而正常幼虫感染则不能诱导较高的抗再感染的效果,提示辐照致弱线虫幼虫来源的细胞或分子具有较好的免疫原性特征,而正常幼虫的则具有耐受原性特征。另外,近年来在抗肿瘤免疫研究中也发现,用某些药物和光辐照疗法处理一些肿瘤细胞后免疫动物,也能够诱导较高水平的抗肿瘤效果,其机制可能是经处理的肿瘤细胞启动了免疫原性细胞死亡程序,触发了特异的信号和效应分子,与宿主先天免疫细胞作用,最终诱导了抗肿瘤的免疫应答。因此,我们推测:辐照致弱线虫幼虫细胞有可能出现免疫原性细胞死亡(坏死性凋亡),能够刺激宿主的保护性免疫应答,而正常线虫幼虫细胞则可能以凋亡为主,主要诱导宿主的免疫耐受。但这一假说是否成立,仍有待我们实验确定。为此,本研究首先对体外培养不同时期的辐照致弱和正常捻转血矛线虫幼虫的生长变化特征及凋亡和坏死进行了比较观察;然后对它们的基因表达谱变化情况进行了分析,旨在为进一步阐明辐照幼虫免疫原性细胞死亡的机制提供依据。首先,我们培养收集捻转血矛线虫三期幼虫,采用紫外辐照方法致弱捻转血矛线虫幼虫。体外培养,光学显微镜观察0d(辐照后2 h),2d,5d,10d,13d和16d不同时期的幼虫的发育情况,设正常幼虫为对照组。结果发现:随着体外培养时间的延长,正常组幼虫形态呈现正常发育特征,而辐照组中,0d时,辐照致弱组幼虫与正常组幼虫形态和活力基本无差异。2d时,两组幼虫都有发生脱鞘的现象,但辐照致弱组脱鞘的比例大于正常组的;两组食道和体表无显著差异,且活力都较好。5d时,两组幼虫均有部分L3发育至L4,辐照致弱组幼虫发育至L4的比例低于正常组幼虫,且辐照组未脱鞘的L3幼虫活力明显降低,活动幅度减少,肠细胞死亡,细胞形态消失。10d时,正常组幼虫约有80%发育至四期,辐照组幼虫却只有20%左右发育率且虫体形态开始发生变化,有的幼虫出现萎缩现象,活力明显减小。13d时,辐照组幼虫体表凹凸不平,肠道颜色加深,虫体皱缩;正常组幼虫活力有所下降,虫体出现萎缩现象。16d时,辐照组幼虫活力消失,体表紊乱,体内有空泡;而大部分正常组幼虫仍然具有活力。说明辐照后的幼虫呈现出明显的类似坏死性凋亡的形态学变化,而正常组幼虫呈现凋亡的形态特征。其次,为了进一步确定辐照捻转血矛线虫幼虫细胞是否出现了坏死性凋亡样现象,培养收集捻转血矛线虫三期幼虫,采用紫外辐照方法致弱捻转血矛线虫幼虫,体外培养至0d,2d,5d,10d,13d和16d,运用扫描电镜(SEM)和透射电镜(TEM)观察幼虫超微结构变化。SEM结果显示:与正常组幼虫比较,辐照组幼虫更早出现体表萎缩,后期体表呈现凹凸不平和扭曲的现象,且萎缩扭曲程度更严重。TEM结果显示,早期两组幼虫未呈现明显差异变化,10-16d正常组幼虫呈现出逐渐凋亡的表型变化,而辐照组幼虫呈现出更明显的坏死性凋亡样表型变化。说明辐照和正常幼虫的确具有上述不同的死亡方式。最后,运用RNA-seq测序技术,对正常组和辐照组捻转血矛线虫幼虫体外培养不同时期的基因表达谱变化进行了分析。发现在培养早期,一些与细胞早期凋亡和生长相关的基因或信号途径高度富集;而在培养晚期,与细胞坏死等相关的基因或途径得到富集。进一步鉴定了3个与免疫原性细胞凋亡相关的基因HSP70、HMG和CRT,发现它们在早期辐照组中的表达量高于正常组的。这些结果为深入探索辐照致弱幼虫免疫原性细胞死亡的分子机制提供了基础。总之,本研究发现辐照捻转血矛线虫幼虫呈现出与坏死性凋亡相关的表型变化,初步探索了其分子机制,鉴定了3个与免疫原性细胞凋亡相关的基因。为进一步阐明辐照幼虫免疫原性细胞死亡的机制奠定了基础。
[Abstract]:Haemonchus contortus wool Yuanke blood spear nematodes, is one of the major gastrointestinal parasites of goats and other ruminant animal, animal disease usually manifested as anemia, weight loss and even death, causing huge economic losses. The main control strategy is chemical anthelmintic drug use, but with the emergence of drug-resistant strains, chemical drug therapy is a serious challenge. People are working hard to seek new prevention strategies, including immunization with R & D molecular vaccine of particular attention, but so far there is a kind of high molecular vaccine protective can be widely used in the field. Previous research found that the application of gamma ray and ultraviolet irradiation attenuated h.contortus the nematode Trichostrongylus colubriformis larvae and immune animals have protective effect was higher, while the normal larvae can not induce higher anti infection effect, suggesting that radiation attenuated nematode larvae The cellular or molecular insect source immunogenic characteristics better, and the larvae have the same characteristic of tolerance. In addition, also found in the study of anti tumor immunity in recent years, with some drugs and radiation therapy in the treatment of some immune animal tumor cells, can induce higher levels of anti-tumor effect the mechanism may be treated tumor cells start the immunogenicity of programmed cell death, triggering signal and specific effector molecules, and the role of the host innate immune cells, induce immune response against tumor. Therefore, we hypothesized that radiation attenuated cell nematode larvae may appear immunogenic cell death (necrosis), apoptosis can stimulate the protective immune response of the host, while normal cells may nematode larvae mainly apoptosis, mainly induced host immune tolerance. But this hypothesis is not established, remains to be We experimentally determined. Therefore, this study first in vitro irradiation in different periods of attenuated and normal Haemonchus contortus larvae growth changes and apoptosis and necrosis were observed; then the changes of gene expression profile were analyzed, in order to provide a basis for further elucidation of the mechanisms of irradiated larvae immunogenicity cell death. Firstly, we collect the cultivation of Haemonchus contortus larvae three, by UV irradiation method attenuated Haemonchus contortus larvae. In vitro, 0d optical microscope (2 h after irradiation), 2D, 5D, 10d, the development of different periods of larvae of 13D and 16d,. Normal larvae as the control group. Results: with the prolongation of culture time, the normal group showed normal morphology of larvae development characteristics, and the irradiation group, 0d group, radiation attenuated larvae and larval morphology and viability of normal group no difference ISO.2d, occurrence exsheath have the phenomenon of two groups of larvae, but radiation attenuated exsheath group ratio is higher than normal group; the two groups had no significant difference between the esophagus and the surface, and the activity of.5d were good, two groups of larvae were part of L3 development to L4, radiation attenuated the growth of larvae to L4 group the proportion is lower than the normal group and the larvae, L3 larvae from the sheath without irradiation group activity decreased obviously, activity decrease, intestinal cell death, cell morphology of.10d disappeared, normal group larvae about 80% to four growth period, the irradiation group larvae was only about 20% growth rate and morphology changes, some larvae emerge the phenomenon of shrinking,.13d activity decreased significantly, the irradiation group on larva is uneven, intestinal color deepens, body shrinkage; normal group activity decreased insect larvae, shrink.16d, irradiation group larvae vitality disappear, skin disorders, and large vacuoles in vivo; Normal group still has vitality. The larvae of irradiated larvae showed similar morphological changes of apoptosis necrosis obviously, while the normal group showed morphological characteristics of larvae apoptosis. Secondly, in order to further determine the irradiated larvae of h.contortus cells appeared necrosis and apoptosis phenomenon, culture collection of Haemonchus contortus the three stage larvae, by ultraviolet irradiation method attenuated Haemonchus contortus larvae cultured in vitro to 0d, 2D, 5D, 10d, 13D and 16d, by using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were observed the ultrastructural changes of.SEM showed that: compared with the normal group of larvae, larvae before irradiation group the surface appears to shrink, later showed uneven and distorted surface phenomenon, and the distortion degree of atrophy is more serious.TEM results showed that two groups of early larvae did not show significant difference, 10-16d group of normal larvae gradually apoptosis Phenotypic changes, and the irradiation group larvae showing necrotic apoptosis like phenotype changes were more obvious. It shows that the irradiation and normal larvae did indeed have the different ways of death. Finally, using RNA-seq sequencing technology, the normal group and the irradiation group of Haemonchus contortus larvae cultured in different periods of gene expression profiles were analyzed. Found in the early stage of culture, and some of the early cell apoptosis and growth of genes or related signal pathways are highly enriched in culture; and late genes or pathways associated with cell necrosis. The enriched further identified 3 primary immune cells and apoptosis related genes HSP70, HMG and CRT, they found higher than the normal group in the early expression quantity in the irradiation group. These results and to explore the molecular mechanism of weak larval immunogenic cell death induced by irradiation provides a basis. In short, the study found that irradiation of twisting The phenotype changes related to necrotic apoptosis were found in the larvae of C. elegans. The molecular mechanism was preliminarily explored, and 3 genes related to immunogenic cell apoptosis were identified. It laid a foundation for further elucidating the mechanism of cell death in irradiated larval immunogenicity.

【学位授予单位】：上海师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.7

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