日本血吸虫正常发育与发育阻遏雌虫形态结构观察和差异miRNAs研究

发布时间：2018-06-05 15:01

本文选题：血吸虫生殖发育 + 差异miRNA　；参考：《中国农业科学院》2015年硕士论文

【摘要】：日本血吸虫正常发育雌虫所产虫卵是造成宿主主要病理损害与疾病再传播的根源,单性感染雌虫不能发育为成熟雌虫,这种发育阻遏雌虫无法在宿主体内正常产卵,因而不会对宿主造成严重的病理损害,也不会造成血吸虫病的流行。鉴于成熟雌虫所产虫卵在病理及疾病传播中的作用,对雌虫生殖系统发育的研究将会为防治血吸虫病理和控制血吸虫再传播有重要意义。为了更进一步探讨雌虫生殖系统发育机制,本研究收集单性感染和混合感染25天后的雌虫,对二者的形态差异、mi RNA差异表达进行了研究,并对其中差异表达显著的let-7进行了初步的功能探讨。一、正常发育与发育阻遏雌虫形态学观察和卵黄腺凋亡水平检测采用激光共聚焦、透射电镜对二者形态进行了仔细的观察。结果表明,发育阻遏雌虫在虫体大小,器官形态,细胞及亚细胞水平与正常雌虫均存在显著的差异。发育阻遏雌虫不仅整个虫体较小,而且生殖器官形态差异显著。在细胞水平上,发育阻遏雌虫卵巢和卵黄腺中没有发现成熟细胞。在亚细胞水平上,成熟雌虫卵黄腺特有脂滴结构也未能在发育阻遏雌虫中观察到。此外,本研究采用TUNEL法对二者的卵黄腺凋亡情况进行了观察,实验结果表明二者卵黄腺细胞的凋亡状况均不明显,均不存在显著的凋亡现象。在实验的过程中,我们观察到正常发育雌虫成形虫卵及卵黄腺细胞所具有的特异性荧光现象,这种特异性荧光存在于虫卵和卵黄腺中,但未在雄虫及未成熟的(混合感染后14天)雌虫中发现,但其物质基础还有待进一步实验证实。二、正常发育与发育阻遏雌虫差异表达mi RNA分析基于二代测序的方法,本研究对正常发育与发育阻遏雌虫差异表达的mi RNA进行了测序分析。本次实验两个样品获得原始reads分别为9358376和9314275个,其中比对上基因组的reads分别为5237397和5244859,比对上已知mi RNA的reads数量分别为2301243和3955847。通过对已知mi RNA差异表达分析,共发现38个显著差异表达的mi RNA,其中在发育阻遏雌虫中上调的有21条(较正常发育雌虫),下调的有17条,并对其中差异表达的mi RNA进行了Real-time PCR验证。表达丰度较高的mi RNA如:mi R-1,mi R-let-7,mi R-124-3p,mi R-36-3p等在发育阻遏雌虫中呈现上调表达的现象。Bantam是唯一高丰度表达mi RNA中,在正常发育雌虫中上调表达的mi RNA。通过生物信息学方法,对这些差异表达的mi RNA进行靶基因预测,再对靶基因进行GO和KEGG分析。GO的分析结果表明,靶基因存在于多个细胞组分中。KEGG分析结果表明细胞粘附,细胞周期相关通路中基因数目较多。三、let-7功能的初步探讨基于二代测序结果,let-7在发育阻遏雌虫中表达量更高,本研究通过对混合感染的小鼠进行注射let-7 mimics,初步探讨其功能。通过Real time-PCR对混合感染后25天的虫体进行let-7的候选靶基因(共挑选18个)验证,以及扫描电镜对体表进行观察,探讨对虫体的影响。结果表明,干扰后虫体的多个候选靶基因明显的下调。对感染42天雌虫的产卵量和虫卵孵化率进行统计,结果表明虫卵孵化率显著上升。对宿主肝脏虫卵肉芽肿的体积进行统计,结果表明实验组肉芽肿体积显著减小。对感染后25天的雌虫进行扫描电镜观察,但各组虫体体表结构无显著差异。
[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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7

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