肺炎衣原体在HEp-2细胞中的发育周期及其感染对细胞迁移的影响

发布时间：2018-06-08 05:25

  本文选题：肺炎衣原体 + 血管平滑肌细胞　； 参考：《天津医科大学》2009年硕士论文

【摘要】： 目的: 研究肺炎衣原体(Chlamdia pneumonia,C.pn)在体外培养中的增殖规律,了解其超微结构的变化,探讨C.pn在体外连续培养的方法;观察C.pn感染大鼠血管平滑肌细胞(vascular smooth muscle cell,VSMC)及人喉癌细胞(HumanEpidermoid Carcinoma-2,HEp-2)后,对细胞迁移能力的改变。 方法: 1.C.pn感染HEp-2细胞,光镜下观察细胞病变,并用PCR技术鉴定其核酸。 2.荧光显微镜下观察感染24h、48h和72h后C.pn在HEp-2细胞内的形态变化。 3.透射电镜下观察感染24h、48h和72h后C.pn在HEp-2细胞内的超微结构变化。 4.应用组织培养半数感染量法测定C.pn感染导致HEp-2细胞病变程度。 5.应用MTT法检测C.pn感染VSMC、HEp-2细胞后,细胞活力的改变。 6.应用MTT法观察C.pn感染对VSMC、HEp-2细胞粘附能力的影响。 7.应用Wound-healing assay和Transwell assay观察C.pn感染对VSMC、HEp-2细胞迁移能力的影响。 结果: 1.在感染24～72h后,光镜下可见HEp-2细胞肿胀、脱落;在感染24h后,包涵体始见于HEp-2细胞胞浆内,至72h占据整个胞浆。PCR扩增出C.pn 437bp的特异性片段。 2.荧光显微镜显示在不同时间点包涵体内由原体向网状体,而后再转变为原体的衍变过程。 3.透射电镜下观察到相似结果,并可见梨形原体及微型小体等C.pn特征性的超微结构。 4.与感染后84h、96h相比,感染后72h收集HEp-2细胞可获得高感染性的子代C.pn。 5.C.pn感染可以显著增强体外培养的VSMC、HEp-2细胞的活力,其最高增殖率分别达到171.54%、43.63%。 6.C.pn感染可显著增加VSMC、HEp-2细胞粘附于Ⅰ型胶原的能力,其最高粘附率分别达到34.4%、19.9%。 7.C.pn感染可明显增强VSMC、HEp-2细胞的迁移能力。 结论: C.pn在HEp-2细胞内经72h完成一个发育周期,感染后72h可获得高感染性的C.pn用于连续传代;C.pn感染能促进VSMC和HEp-2细胞迁移。
[Abstract]:Objective: to study the proliferation of chlamdia pneumoniae C. pnn in vitro, to investigate the ultrastructural changes of C. pn, to explore the method of continuous culture of C. pn in vitro, to observe the infection of C. pn in vascular smooth muscle cells (VSMC) of rats and human laryngeal carcinoma cell line human Epidermoid Carcinoma-2HEp-2). Methods: 1. HEp-2 cells were infected with C.pn. The pathological changes were observed under light microscope, and the nucleic acids of HEp-2 cells were identified by polymerase chain reaction (PCR). 2. The morphological changes of C. pn in HEp-2 cells were observed under fluorescence microscope for 48 h and 72 h after infection. The ultrastructural changes of C.pn in HEp-2 cells were observed under transmission electron microscope for 48 h and 72 h after infection. 4. The pathological degree of HEp-2 cells induced by C.pn infection was determined by the method of the half infective dose of tissue culture. MTT assay was used to detect the changes of cell viability of VSMC- Hep-2 cells infected with C. pn. MTT assay was used to observe the effect of C.pn infection on adhesion ability of VSMC- HEp-2 cells. Wound-healing assay and Transwell assay were used to observe the effect of C.pn infection on the migration ability of VSMC-HEp-2 cells. After 24 hours of infection, HEp-2 cells were swollen and shedded under light microscope, and the inclusion bodies were first found in the cytoplasm of HEp-2 cells at 24 h after infection, and the specific fragment of C.pn 437bp was amplified at 72 h after infection and occupied the whole cytoplasm of HEp-2 cells. The fluorescence microscope showed the evolution of inclusion body from the plasma to the reticular body at different time points, and then to the evolution of the plasma. 3. Similar results were observed under transmission electron microscope, and the characteristic ultrastructure of C. pn such as Pyridia and microcorpuscles was observed. 4. Compared with 84 h after infection and 96 h after infection, the highly infectious progeny C.pn.5.C.pn infection could significantly enhance the activity of VSMC-HEp-2 cells collected 72 h after infection. The highest proliferative rate was 171.54 and 43.63.The infection of C.pn significantly increased the ability of VSMCS-HEp-2 cells to adhere to type 鈪，

本文编号：1994703