本文選題：血吸蟲生殖發(fā)育 + 差異miRNA　； 參考：《中國農(nóng)業(yè)科學院》2015年碩士論文

【摘要】：日本血吸蟲正常發(fā)育雌蟲所產(chǎn)蟲卵是造成宿主主要病理損害與疾病再傳播的根源,單性感染雌蟲不能發(fā)育為成熟雌蟲,這種發(fā)育阻遏雌蟲無法在宿主體內(nèi)正常產(chǎn)卵,因而不會對宿主造成嚴重的病理損害,也不會造成血吸蟲病的流行。鑒于成熟雌蟲所產(chǎn)蟲卵在病理及疾病傳播中的作用,對雌蟲生殖系統(tǒng)發(fā)育的研究將會為防治血吸蟲病理和控制血吸蟲再傳播有重要意義。為了更進一步探討雌蟲生殖系統(tǒng)發(fā)育機制,本研究收集單性感染和混合感染25天后的雌蟲,對二者的形態(tài)差異、mi RNA差異表達進行了研究,并對其中差異表達顯著的let-7進行了初步的功能探討。一、正常發(fā)育與發(fā)育阻遏雌蟲形態(tài)學觀察和卵黃腺凋亡水平檢測采用激光共聚焦、透射電鏡對二者形態(tài)進行了仔細的觀察。結(jié)果表明,發(fā)育阻遏雌蟲在蟲體大小,器官形態(tài),細胞及亞細胞水平與正常雌蟲均存在顯著的差異。發(fā)育阻遏雌蟲不僅整個蟲體較小,而且生殖器官形態(tài)差異顯著。在細胞水平上,發(fā)育阻遏雌蟲卵巢和卵黃腺中沒有發(fā)現(xiàn)成熟細胞。在亞細胞水平上,成熟雌蟲卵黃腺特有脂滴結(jié)構(gòu)也未能在發(fā)育阻遏雌蟲中觀察到。此外,本研究采用TUNEL法對二者的卵黃腺凋亡情況進行了觀察,實驗結(jié)果表明二者卵黃腺細胞的凋亡狀況均不明顯,均不存在顯著的凋亡現(xiàn)象。在實驗的過程中,我們觀察到正常發(fā)育雌蟲成形蟲卵及卵黃腺細胞所具有的特異性熒光現(xiàn)象,這種特異性熒光存在于蟲卵和卵黃腺中,但未在雄蟲及未成熟的(混合感染后14天)雌蟲中發(fā)現(xiàn),但其物質(zhì)基礎(chǔ)還有待進一步實驗證實。二、正常發(fā)育與發(fā)育阻遏雌蟲差異表達mi RNA分析基于二代測序的方法,本研究對正常發(fā)育與發(fā)育阻遏雌蟲差異表達的mi RNA進行了測序分析。本次實驗兩個樣品獲得原始reads分別為9358376和9314275個,其中比對上基因組的reads分別為5237397和5244859,比對上已知mi RNA的reads數(shù)量分別為2301243和3955847。通過對已知mi RNA差異表達分析,共發(fā)現(xiàn)38個顯著差異表達的mi RNA,其中在發(fā)育阻遏雌蟲中上調(diào)的有21條(較正常發(fā)育雌蟲),下調(diào)的有17條,并對其中差異表達的mi RNA進行了Real-time PCR驗證。表達豐度較高的mi RNA如:mi R-1,mi R-let-7,mi R-124-3p,mi R-36-3p等在發(fā)育阻遏雌蟲中呈現(xiàn)上調(diào)表達的現(xiàn)象。Bantam是唯一高豐度表達mi RNA中,在正常發(fā)育雌蟲中上調(diào)表達的mi RNA。通過生物信息學方法,對這些差異表達的mi RNA進行靶基因預測,再對靶基因進行GO和KEGG分析。GO的分析結(jié)果表明,靶基因存在于多個細胞組分中。KEGG分析結(jié)果表明細胞粘附,細胞周期相關(guān)通路中基因數(shù)目較多。三、let-7功能的初步探討基于二代測序結(jié)果,let-7在發(fā)育阻遏雌蟲中表達量更高,本研究通過對混合感染的小鼠進行注射let-7 mimics,初步探討其功能。通過Real time-PCR對混合感染后25天的蟲體進行l(wèi)et-7的候選靶基因(共挑選18個)驗證,以及掃描電鏡對體表進行觀察,探討對蟲體的影響。結(jié)果表明,干擾后蟲體的多個候選靶基因明顯的下調(diào)。對感染42天雌蟲的產(chǎn)卵量和蟲卵孵化率進行統(tǒng)計,結(jié)果表明蟲卵孵化率顯著上升。對宿主肝臟蟲卵肉芽腫的體積進行統(tǒng)計,結(jié)果表明實驗組肉芽腫體積顯著減小。對感染后25天的雌蟲進行掃描電鏡觀察,但各組蟲體體表結(jié)構(gòu)無顯著差異。
[Abstract]:The egg produced by the normal development of the female Schistosoma japonicum is the source of the main pathological damage of the host and the retransmission of the disease. The single sex infected female can not develop into a mature female. This depressor is not able to spawn normally in the host. Therefore, it does not cause serious damage to the host and does not cause the epidemic of schistosomiasis. The role of the eggs produced by mature females in the spread of pathology and disease and the study of reproductive system development of the female will be of great significance for the prevention and control of the pathology of schistosomiasis and the control of the re transmission of Schistosoma. In order to further explore the developmental mechanism of the reproductive system of the female, this study collects 25 days after a single infection and a mixed infection, to two The morphological differences and the differential expression of MI RNA were studied, and a preliminary functional discussion was carried out on the significant differences in expression of let-7. First, the morphological observation of the normal development and development repression of the female and the detection of the level of the yolk gland apoptosis were examined by laser confocal microscopy, and the transmission electron microscope was carefully observed for the two forms. There were significant differences in the size, organ morphology, cell and subcellular level of the depressant females. The developmental repression was not only small, but also the morphological differences of the reproductive organs were significant. At the cell level, the mature cells were not found in the ovary and the yolk gland of the depressor. At the subcellular level, the mature females were at the level of subcellular level. The specific lipid droplet structure of the yolk gland has not been observed in the developmental repression female. In addition, the TUNEL method was used to observe the apoptosis of the yolk gland in two cases. The experimental results showed that the apoptotic status of the two yolk gland cells was not obvious, and there was no significant apoptosis. In the process of the experiment, we observed normal development. The specific fluorescence phenomenon of the egg and the yolk gland cells of the female insect is found in the egg and the yolk gland, but it is not found in the male and the immature (14 days after the mixed infection), but the material basis remains to be confirmed by the further experiment. Two, the difference expression of the normal development and development repression of the female is mi RNA. Based on the two generation sequencing method, the MI RNA of the differential expression of normal development and development repressor was sequenced in this study. The two samples of this experiment obtained 9358376 and 9314275 original reads respectively, of which the reads of the comparison of the upper genome was 5237397 and 5244859, respectively, and the reads number of the MI RNA was 230124, respectively, 230124. 3 and 3955847. through the analysis of the differential expression of known mi RNA, 38 significant differentially expressed mi RNA were found, of which 21 (more normal developing females) were up-regulated in the developmental repressor and 17 down regulated, and the MI RNA of the differentially expressed mi RNA was tested by Real-time PCR. The up-regulated expression of 124-3p, MI R-36-3p and so on in the depressor female.Bantam is the only high abundance expression of MI RNA. The MI RNA., up regulated in the normal developing female, is predicted by the Bioinformatics Method and the target gene is predicted for these differentially expressed mi RNA, and the analysis results of GO and KEGG analysis of the target genes are shown. .KEGG analysis of the target gene in multiple cell components showed that the cell adhesion and the number of genes in the cell cycle related pathway were more. Three, the preliminary study on the function of let-7 was based on the results of the two generation sequencing, and the expression of let-7 in the depressor female was higher. This study was conducted by injecting let-7 mimics into the mixed infected mice. The function. Through Real time-PCR, the candidate target gene of let-7 (a total of 18) for the 25 days after mixed infection was tested, and the effect on the body was investigated by scanning electron microscope to observe the body surface. The results showed that the egg hatching rate was significantly increased. The volume of the egg granuloma in the liver of the host was statistically analyzed. The results showed that the granuloma volume decreased significantly in the experimental group. There was a scanning electron microscope for the female 25 days after infection, but there was no significant difference in the body surface structure of each group.
【學位授予單位】：中國農(nóng)業(yè)科學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.7

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