曼氏裂头蚴感染诊断的病原鉴定方法研究

发布时间：2018-05-29 08:26

本文选题：曼氏裂头蚴 + 病原诊断　；参考：《中南大学》2012年硕士论文

【摘要】：[目的]系统观察和分析曼氏裂头蚴的解剖组织学和组织病理学用以补充对该病原诊断基础的不足；探索检测切片中曼氏裂头蚴抗原和基因的方法用以解决病理切片中不能确定虫种的鉴定技术问题。 [方法]收集来自青蛙和病人的曼氏裂头蚴标本42个、来自动物的其他蠕虫标本13个和人体组织标本2个。用倒置显微镜比较观察裂头蚴各部位的解剖结构组织学特征；福尔马林固定和石蜡包埋的曼氏裂头蚴切片标本,对HE染色的虫体不同部位组织学进行比较观察；制备曼氏裂头蚴免疫血清,用间接酶免疫组化法比较检测不同虫种切片组织中抗原；设计作曼氏裂头蚴mtDNACOX1基因部分片断检测的引物4对,用商品化试剂分别提取数种蠕虫基因组DNA作PCR,取扩增产物经电泳观察目的条带,并测序,用blast软件分析其与已知基因序列的符合率。 [结果]曼氏裂头蚴显微解剖组织学特征为：头部吸槽内具囊腔；体壁具凹凸横皱褶；体内实质呈网状纤维结构,富含石灰小体,在体中后部可见排泄小管；从虫体划破处可见有大量的呈圆形或椭圆形、无细胞结构的强折光盘状体溢出,即石灰小体,其个体明显比体细胞大。曼氏裂头蚴组织病理学特征为：可因切取部位不同而有差异；头部形态最不规则,其横切面可见囊腔和成石灰小体；体部和尾部断面较大；体部的不同切面在组织结构上有差异；除头部外,虫体各断面的共同特征为体壁具凹凸不等的横皱褶,皮层较厚呈嗜伊红深染,有的皮层外可见长短不一微毛,体内为网状疏松的实质组织,无体腔和消化器官结构,可见分散的肌纤维和圆形,椭圆形或不规则形石灰小体,亦可见排泄管腔；在病理切片9例中有5例在虫体组织内未见典型的网状结构(呈钙化变性),石灰小体和肌纤维束结构不典型。用酶免疫组化法最佳条件,对已知并殖吸虫、猪囊尾蚴和曼氏裂头蚴的组织切片检测均未出现假阴性和假阳性,对12例临床来源的病理切片中裂头蚴检测均呈阳性反应。在基因扩增用的4对引物中,有两对(F650/R800和F965/R1120)可检出曼氏裂头蚴特异性目的条带。 [结论]本研究描述的曼氏裂头蚴显微解剖组织学特征与其观察方法为病理学对石蜡包埋前虫体鉴定提供了简捷手段；系统观察和阐述的裂头蚴组织病理学特征为病理学观察提供了参考依据；探索检测曼氏裂头蚴特异性抗原和基因的方法为病理切片中疑似虫体鉴定提供了技术。
[Abstract]:[objective] to observe and analyze systematically the anatomical histology and histopathology of caterpillars mansoni in order to supplement the deficiency of the basis of diagnosis of the pathogen. To explore the method of detecting antigen and gene of mitomycaria mansoni in order to solve the problem of identification of insect species in pathological sections. Methods 42 specimens of mitomycaria mansoni from frogs and patients, 13 specimens of other worms from animals and 2 specimens of human tissues were collected. The anatomical structure and histological characteristics of each part of the caterpillar were observed by inverted microscope, and the histological characteristics of the different parts of the insect body stained by HE were compared with those of the specimens fixed with formalin and paraffin embedded in the sections of the filariae. To prepare the immune serum of mitomycaria mansoni, to compare and detect the antigens in the sections of different insect species by indirect enzyme immunohistochemical method, and to design 4 pairs of primers for the partial detection of mtDNACOX1 gene fragment of mitomycaria mansoni. Several worm genomic DNA were extracted by commercial reagents as PCR products. The target bands were observed by electrophoresis and sequenced. The coincidence rate of the PCR products with known gene sequences was analyzed by blast software. [results] the microanatomical and histological features of the caterpillar were as follows: there was a cystic cavity in the suction groove of the head, a convoluted transverse fold in the body wall, a reticular fibrous structure in the body, rich in lime bodies, and a excretory tubule in the posterior part of the body. A large number of strongly refractive disklike bodies with circular or elliptical shape and no cell structure were found to overflow from the area of the insect body, that is, calcareous bodies, whose individual size was obviously larger than that of somatic cells. The histopathological features of mitocercariae mansoni are as follows: the shape of the head is the most irregular, the cystic cavity and calcareous bodies can be seen on the transverse section of the head, the section of body and tail is large. In addition to the head, the common features of the different sections of the insect body are the uneven transverse folds of the body wall, the thicker layer of the body and the deep staining of the iridescent, and some of them have different hairs of different lengths outside of the cortex, and the common features of the different sections of the body are that the body wall has different transverse folds, and the cortical layer is thicker and reddish. The body was reticular-loose parenchyma, without the structure of body cavity and digestive organ, with scattered muscle fiber and circular, elliptic or irregular calcareous bodies, and also with excretory lumen. Typical reticular structures (calcified denaturation, calcareous bodies and muscular fiber bundles) were not found in 5 of 9 cases of pathological sections. Under the best condition of enzyme immunohistochemical method, no false negative and false positive staining was found in the sections of known paragonimiasis, cysticercus and cysticercus mansoni, but positive reaction was found in 12 pathological sections of clinical origin. Of the 4 pairs of primers used for gene amplification, two pairs of F650 / R800 and F965 / R1120) could detect the specific target bands of mitomycaria mansoni. [conclusion] the microanatomical and histological characteristics and observation methods described in this study provide a simple and convenient method for the identification of paraffin embedded prebugs by pathology. The histopathological characteristics of mitomycaria provided a reference basis for pathological observation, and a method for detecting specific antigen and gene of mitomycaria mansoni provided a technique for identification of suspected parasites in pathological sections.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R383.35

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