本文關(guān)鍵詞： 捻轉(zhuǎn)血矛線蟲 輻照致弱幼蟲 免疫原性細(xì)胞死亡 凋亡　出處：《上海師范大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：捻轉(zhuǎn)血矛線蟲屬毛圓科血矛屬線蟲,是山羊等反芻動物的主要胃腸道寄生蟲之一,患病動物通常表現(xiàn)為貧血、消瘦甚至死亡,造成巨大經(jīng)濟(jì)損失。目前的主要防治方法是使用化學(xué)藥物驅(qū)蟲,但隨著抗藥性蟲株的的出現(xiàn),化學(xué)藥品治療正受到嚴(yán)重的挑戰(zhàn)。人們正在努力尋求新的防控策略,其中免疫預(yù)防用分子疫苗的研發(fā)尤其受到重視,但迄今沒有一種高保護(hù)性的分子疫苗能夠推廣應(yīng)用于現(xiàn)場。以前的研究發(fā)現(xiàn),應(yīng)用γ射線和紫外線照射致弱的捻轉(zhuǎn)血矛線蟲和蛇形毛圓線蟲幼蟲免疫家畜都獲得了較高的保護(hù)性效果,而正常幼蟲感染則不能誘導(dǎo)較高的抗再感染的效果,提示輻照致弱線蟲幼蟲來源的細(xì)胞或分子具有較好的免疫原性特征,而正常幼蟲的則具有耐受原性特征。另外,近年來在抗腫瘤免疫研究中也發(fā)現(xiàn),用某些藥物和光輻照療法處理一些腫瘤細(xì)胞后免疫動物,也能夠誘導(dǎo)較高水平的抗腫瘤效果,其機(jī)制可能是經(jīng)處理的腫瘤細(xì)胞啟動了免疫原性細(xì)胞死亡程序,觸發(fā)了特異的信號和效應(yīng)分子,與宿主先天免疫細(xì)胞作用,最終誘導(dǎo)了抗腫瘤的免疫應(yīng)答。因此,我們推測:輻照致弱線蟲幼蟲細(xì)胞有可能出現(xiàn)免疫原性細(xì)胞死亡(壞死性凋亡),能夠刺激宿主的保護(hù)性免疫應(yīng)答,而正常線蟲幼蟲細(xì)胞則可能以凋亡為主,主要誘導(dǎo)宿主的免疫耐受。但這一假說是否成立,仍有待我們實驗確定。為此,本研究首先對體外培養(yǎng)不同時期的輻照致弱和正常捻轉(zhuǎn)血矛線蟲幼蟲的生長變化特征及凋亡和壞死進(jìn)行了比較觀察;然后對它們的基因表達(dá)譜變化情況進(jìn)行了分析,旨在為進(jìn)一步闡明輻照幼蟲免疫原性細(xì)胞死亡的機(jī)制提供依據(jù)。首先,我們培養(yǎng)收集捻轉(zhuǎn)血矛線蟲三期幼蟲,采用紫外輻照方法致弱捻轉(zhuǎn)血矛線蟲幼蟲。體外培養(yǎng),光學(xué)顯微鏡觀察0d(輻照后2 h),2d,5d,10d,13d和16d不同時期的幼蟲的發(fā)育情況,設(shè)正常幼蟲為對照組。結(jié)果發(fā)現(xiàn):隨著體外培養(yǎng)時間的延長,正常組幼蟲形態(tài)呈現(xiàn)正常發(fā)育特征,而輻照組中,0d時,輻照致弱組幼蟲與正常組幼蟲形態(tài)和活力基本無差異。2d時,兩組幼蟲都有發(fā)生脫鞘的現(xiàn)象,但輻照致弱組脫鞘的比例大于正常組的;兩組食道和體表無顯著差異,且活力都較好。5d時,兩組幼蟲均有部分L3發(fā)育至L4,輻照致弱組幼蟲發(fā)育至L4的比例低于正常組幼蟲,且輻照組未脫鞘的L3幼蟲活力明顯降低,活動幅度減少,腸細(xì)胞死亡,細(xì)胞形態(tài)消失。10d時,正常組幼蟲約有80%發(fā)育至四期,輻照組幼蟲卻只有20%左右發(fā)育率且蟲體形態(tài)開始發(fā)生變化,有的幼蟲出現(xiàn)萎縮現(xiàn)象,活力明顯減小。13d時,輻照組幼蟲體表凹凸不平,腸道顏色加深,蟲體皺縮;正常組幼蟲活力有所下降,蟲體出現(xiàn)萎縮現(xiàn)象。16d時,輻照組幼蟲活力消失,體表紊亂,體內(nèi)有空泡;而大部分正常組幼蟲仍然具有活力。說明輻照后的幼蟲呈現(xiàn)出明顯的類似壞死性凋亡的形態(tài)學(xué)變化,而正常組幼蟲呈現(xiàn)凋亡的形態(tài)特征。其次,為了進(jìn)一步確定輻照捻轉(zhuǎn)血矛線蟲幼蟲細(xì)胞是否出現(xiàn)了壞死性凋亡樣現(xiàn)象,培養(yǎng)收集捻轉(zhuǎn)血矛線蟲三期幼蟲,采用紫外輻照方法致弱捻轉(zhuǎn)血矛線蟲幼蟲,體外培養(yǎng)至0d,2d,5d,10d,13d和16d,運(yùn)用掃描電鏡(SEM)和透射電鏡(TEM)觀察幼蟲超微結(jié)構(gòu)變化。SEM結(jié)果顯示:與正常組幼蟲比較,輻照組幼蟲更早出現(xiàn)體表萎縮,后期體表呈現(xiàn)凹凸不平和扭曲的現(xiàn)象,且萎縮扭曲程度更嚴(yán)重。TEM結(jié)果顯示,早期兩組幼蟲未呈現(xiàn)明顯差異變化,10-16d正常組幼蟲呈現(xiàn)出逐漸凋亡的表型變化,而輻照組幼蟲呈現(xiàn)出更明顯的壞死性凋亡樣表型變化。說明輻照和正常幼蟲的確具有上述不同的死亡方式。最后,運(yùn)用RNA-seq測序技術(shù),對正常組和輻照組捻轉(zhuǎn)血矛線蟲幼蟲體外培養(yǎng)不同時期的基因表達(dá)譜變化進(jìn)行了分析。發(fā)現(xiàn)在培養(yǎng)早期,一些與細(xì)胞早期凋亡和生長相關(guān)的基因或信號途徑高度富集;而在培養(yǎng)晚期,與細(xì)胞壞死等相關(guān)的基因或途徑得到富集。進(jìn)一步鑒定了3個與免疫原性細(xì)胞凋亡相關(guān)的基因HSP70、HMG和CRT,發(fā)現(xiàn)它們在早期輻照組中的表達(dá)量高于正常組的。這些結(jié)果為深入探索輻照致弱幼蟲免疫原性細(xì)胞死亡的分子機(jī)制提供了基礎(chǔ)�？傊�,本研究發(fā)現(xiàn)輻照捻轉(zhuǎn)血矛線蟲幼蟲呈現(xiàn)出與壞死性凋亡相關(guān)的表型變化,初步探索了其分子機(jī)制,鑒定了3個與免疫原性細(xì)胞凋亡相關(guān)的基因。為進(jìn)一步闡明輻照幼蟲免疫原性細(xì)胞死亡的機(jī)制奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：上海師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.7

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