本文選題：肺炎衣原體 + 血管平滑肌細(xì)胞�。� 參考：《天津醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 目的: 研究肺炎衣原體(Chlamdia pneumonia,C.pn)在體外培養(yǎng)中的增殖規(guī)律,了解其超微結(jié)構(gòu)的變化,探討C.pn在體外連續(xù)培養(yǎng)的方法;觀察C.pn感染大鼠血管平滑肌細(xì)胞(vascular smooth muscle cell,VSMC)及人喉癌細(xì)胞(HumanEpidermoid Carcinoma-2,HEp-2)后,對(duì)細(xì)胞遷移能力的改變。 方法: 1.C.pn感染HEp-2細(xì)胞,光鏡下觀察細(xì)胞病變,并用PCR技術(shù)鑒定其核酸。 2.熒光顯微鏡下觀察感染24h、48h和72h后C.pn在HEp-2細(xì)胞內(nèi)的形態(tài)變化。 3.透射電鏡下觀察感染24h、48h和72h后C.pn在HEp-2細(xì)胞內(nèi)的超微結(jié)構(gòu)變化。 4.應(yīng)用組織培養(yǎng)半數(shù)感染量法測(cè)定C.pn感染導(dǎo)致HEp-2細(xì)胞病變程度。 5.應(yīng)用MTT法檢測(cè)C.pn感染VSMC、HEp-2細(xì)胞后,細(xì)胞活力的改變。 6.應(yīng)用MTT法觀察C.pn感染對(duì)VSMC、HEp-2細(xì)胞粘附能力的影響。 7.應(yīng)用Wound-healing assay和Transwell assay觀察C.pn感染對(duì)VSMC、HEp-2細(xì)胞遷移能力的影響。 結(jié)果: 1.在感染24～72h后,光鏡下可見(jiàn)HEp-2細(xì)胞腫脹、脫落;在感染24h后,包涵體始見(jiàn)于HEp-2細(xì)胞胞漿內(nèi),至72h占據(jù)整個(gè)胞漿。PCR擴(kuò)增出C.pn 437bp的特異性片段。 2.熒光顯微鏡顯示在不同時(shí)間點(diǎn)包涵體內(nèi)由原體向網(wǎng)狀體,而后再轉(zhuǎn)變?yōu)樵w的衍變過(guò)程。 3.透射電鏡下觀察到相似結(jié)果,并可見(jiàn)梨形原體及微型小體等C.pn特征性的超微結(jié)構(gòu)。 4.與感染后84h、96h相比,感染后72h收集HEp-2細(xì)胞可獲得高感染性的子代C.pn。 5.C.pn感染可以顯著增強(qiáng)體外培養(yǎng)的VSMC、HEp-2細(xì)胞的活力,其最高增殖率分別達(dá)到171.54%、43.63%。 6.C.pn感染可顯著增加VSMC、HEp-2細(xì)胞粘附于Ⅰ型膠原的能力,其最高粘附率分別達(dá)到34.4%、19.9%。 7.C.pn感染可明顯增強(qiáng)VSMC、HEp-2細(xì)胞的遷移能力。 結(jié)論: C.pn在HEp-2細(xì)胞內(nèi)經(jīng)72h完成一個(gè)發(fā)育周期,感染后72h可獲得高感染性的C.pn用于連續(xù)傳代;C.pn感染能促進(jìn)VSMC和HEp-2細(xì)胞遷移。
[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，

本文編號(hào)：1994703