卡氏肺孢子虫生长发育的观察研究

【作者】 姜云霞；

【导师】 宫玉香；

【作者基本信息】 青岛大学 ， 病原生物学， 2008， 硕士

【摘要】 目的观察Wistar大鼠肺组织中卡氏肺孢子虫的生长发育和形态结构。方法通过皮下注射地塞米松磷酸钠建立Wistar大鼠卡氏肺孢子虫肺炎动物模型为实验组,另设不注射药物为对照组。制备大鼠肺印片标本,瑞-姬氏染色,光学显微镜下检测和观察卡氏肺孢子虫。取大鼠肺组织标本,制备肺组织切片和透射、扫描电镜观察标本,分别使用光镜、透射和扫描电镜观察该虫生长发育、形态结构及其感染大鼠肺病理学变化。用数字图像分析技术,对卡氏肺孢子虫包囊进行形态学参数测定。结果肺印片标本经瑞-姬氏染色,光学显微镜油镜下检测,可见清晰卡氏肺孢子虫包囊和滋养体,以肺泡内多见,偶可在细胞内见到。对照组大鼠未检测到该虫。实验组大鼠肺印片标本该虫检出率为57%,第4～5周查到包囊为主,7～8周后以滋养体为主。扫描电镜观察,卡氏肺孢子虫有表面光滑和粗糙两种类型,其中粗糙型又有微绒毛、管状结构、颗粒样物三种情况;观察到壁上有凹陷、正在脱囊及伸出伪足的虫体。透射电镜观察到卡氏肺孢子虫有滋养体、囊前期和包囊三个发育阶段。滋养体多附着于肺Ⅰ型上皮细胞,偶见附着于肺Ⅱ型上皮细胞,有的伸出1或多个较宽大伪足,部分细胞质内发现核相关细胞器和纺锤微管。囊前期有早、中、晚三种类型,在其内发现有代表减数分裂的联会复合体。包囊壁上有一增厚处,内有一孔隙。第10周实验组大鼠肺间隔及气管中可见到卡氏肺孢子虫。用数字图像处理技术测量卡氏肺孢子虫包囊长径、短径、投影面积、体积、周长、等效直径和圆球度数值均数分别为:4.8159μm,3.6923μm,13.07μm~2,36.76μm~3,14.29μm,4.03μm和0.8。光镜观察HE染色的肺组织切片,第7～8周实验组大鼠肺泡内被大量虫体和渗出物充满,肺Ⅱ型上皮细胞脱落,肺泡隔增厚。扫描电镜观察肺内有卡氏肺孢子虫,肺组织呈纤维性改变。透射电镜观察,肺Ⅱ型上皮细胞质内可见许多空泡和电子密度高的颗粒。含虫体的肺Ⅱ型上皮细胞表膜突出,形成质膜小泡,细胞核膜内陷,核内染色体呈丝状并绕成团。结论瑞-姬氏复染法检测卡氏肺孢子虫,操作简单、虫体清晰,有利于一般实验室对该虫检测。扫描电镜观察到表面有粗细不等的管状物和正在脱囊的虫体,阳性大鼠肺组织纤维化改变,为国内首次报道。透射电镜观察到卡氏肺孢子虫的滋养体、囊前期和包囊三个发育阶段;滋养体附着于肺Ⅱ型上皮细胞及气管内,国内外未见报道;包囊壁上观察到一增厚处,内有一孔隙,为罕见报道。数字图像分析技术获得了卡氏肺孢子虫包囊的一系列数据,丰富了肺孢子虫生物学资料,为病原学诊断提供参考资料。更多还原

【Abstract】 Objective To observe the growth , development and morphological structure of Pneumocystis Carinii in the lung tissue of Wistar ratsMethod The experimental group was consisted of Wistar rat models of Pneumocystis Carinii pneumonia established by subcutaneous injection of dexamethasone sodium phosphate. No injection was given to the rats in the control group. The impression preparations of the lung tissue were processed by Wright-Giemsa stain. Optic microscope was used to detect and observe Pneumocystis Carinii. Slices and specimens for transmission electron microscope and scanning electron microscope were made from the samples of lung tissue. Optic microscope, transmission electron microscope and scanning electron microscope were used for the observation of the growth, development and morphological structure of Pneumocystis Carinii as well as the pathological change of the lung.tissues of the infected rats. Digital image analytic technique was used to measure the various morphological indexes of the Pneumocystis Carinii cysts.Result Optic microscope with oil immersion lens was used to examine the impression preparations of the lung tissue processed by Wright-Giemsa stain. The cysts and trophozoites were clearly observed, more often in the pulmonary alveoli while rarely in the cells. No Pneumocystis was detected in the control group. The positive detection rate was 57.14% in the experimental group. Cysts became the main observation from week 4-5, while trophozoites were the main observation after week 7-8. Under the scanning electron microscope, the Pneumocystis Carinii manifested as either smooth-surfaced or rough-surfaced. Three conditions were observed in the rough-surfaced: dense microvilli, tubular structures and granular substances. Also observed were polypides with dent in the wall and in the process of excystation or extending the pseudopodium. Under the transmission electron microscope, three developmental stages were observed: trophozoite, precyst and cyst. Most trophozoites were observed to attach to type I pulmonary epithelial cells. Their attachment to Type II pulmonary epithelial cells was occasionally seen. Some of them extended one or more comparatively wider Pseudopodia. Nucleolus related organelles and spindle microtubules were found in some cytoplasm. The precyst phase was in three forms: early, intermediate and late form and synaptonemal complexes indicating meiosis were found in precyst. One thickening portion with a pore was observed in the wall of the cyst. In week 10, Pneumocystis Carinii can be seen in the pulmonary septum and trachea in the experimental rats. The cysts of the Pneumocystis Carinii were measured by digital image processing techniques. The mean numerical value of the major diameter, minor diameter, projected area, volume, circumference, equivalent diameter and sphericity was 4.8159μm, 3.6923μm,13.07μm~2 ,36.76μm~3 ,14.29μm,4.03μm and 0.8 respectively. HE stained slices were observed under optic microscope. In week 7-8, the pulmonary alveoli of the experimental rats were filled with a large quantity of polypides and exudates. Type II pulmonary cells were found in exfoliation. Thickening of the interalveolar septum was noticed. Under transmission electron microscope, Pneumocystis Carinii was seen in the lung tissues in which fibrous change was observed. Under transmission electron microscope, a lot of vacuoles and electron-dense granules were seen in the cytoplasm of type II pulmonary epithelial cells. In the infected Type II pulmonary epithelial cells with polypides, the cell pellicle protruded and formed plasmalemma alveolus. The nucleolus membrane was invaginated and the chromosome was fiber-like and circumvolated.Conclusion Using Wright-Giemsa re-stains method for the detection of Pneumocystis Carinii has the advantages of easy operation, and clear observation of the polypides. It is ideal for the detection of Pneumocystis Carinii in common laboratories. We are the first in China to report the observation that there are some tubers on the surface of body and in the process of excystation and fibrosis lung tissue in positive rat under scanning electron microscope. As far as we know, there has been no report home or abroad either on the observation of the three developmental stages, trophozoite, precyst and cyst, of Pneumocystis Carinii under transmission electron microscope, or on the finding of the attachment of trophozoites to type II pulmonary epithelial cells and tracheas. There are fewer reports on the observation of a thickening portion with a pore in the wall of the cyst. Data about Pneumocystis Carinii cysts, which are acquired by digital image analytic technique, amplify biological data of Pneumocystis Carinii and provide the reference for etiological diagnosis.更多还原