本文選題：鼠衣原體　切入點：質粒　出處：《南華大學》2015年博士論文　論文類型：學位論文

【摘要】：衣原體是一類嚴格真核細胞內寄生、具有獨特發(fā)育周期的原核細胞型微生物,可引起人和動物多種疾病,疾病表現(xiàn)復雜。對人致病的衣原體有沙眼衣原體、肺炎衣原體和鸚鵡熱衣原體等。衣原體感染已成為當今世界具有挑戰(zhàn)性的重大公共衛(wèi)生問題和社會問題,對健康和經濟有著巨大的影響。為有效應對衣原體感染給公共衛(wèi)生所帶來的挑戰(zhàn),加強衣原體基礎研究,深入研究其致病機制、研制衣原體疫苗成為世界范圍內衣原體研究學者的重要研究課題。目前,鼠衣原體作為一種模式菌已廣泛應用于人類病原性衣原體致病機制研究和衣原體疫苗研究中。一、鼠衣原體質粒缺失株的構建和鑒定背景與目的:衣原體質粒在衣原體致病中發(fā)揮重要作用,利用質粒缺失菌株和質粒攜帶菌株進行比較研究,可以觀察研究質粒的遺傳學功能,此外質粒缺失菌株還可以用作基因工程菌,廣泛應用于分子生物學研究;因此構建和鑒定衣原體質粒缺失株具有重要意義。方法:采用新生霉素處理鼠衣原體、噬斑形成實驗結合以Pgp3為檢測標志的間接免疫熒光分析方法篩選鼠衣原體質粒缺失株,進一步采用PCR檢測質�；�,碘染色觀察糖原聚集現(xiàn)象,動物試驗檢測其致病性。結果:在96孔微孔板中進行的篩選試驗中,篩選到5個質粒缺失菌株。通過PCR分析證實這些菌株缺乏質粒基因,此外,在上述菌株感染的HeLa細胞中沒有發(fā)現(xiàn)GlgA蛋白的分泌現(xiàn)象,也無糖原聚集。質粒缺失菌株CMUT3經陰道感染C3H/HeJ小鼠未見誘導小鼠輸卵管積水。結論:成功構建了鼠衣原體質粒缺失株。二、體外傳代對鼠衣原體生物學特性和致病性的影響背景與目的:盡管鼠衣原體在實驗室已傳代數十年,有關傳代選擇作用于鼠衣原體的結局尚未見報道。最近,我們對鼠衣原體質粒缺失株cmut3和野生型菌株進行傳代培養(yǎng),采用“無輔助感染和輔助感染”交替的方式進行體外傳代培養(yǎng),然后觀察傳代的菌株感染細胞時對離心輔助感染因素的依賴性;傳代的cmg28和cmut3g40在感染hela細胞時對離心這種輔助感染因素的依賴性降低。c3h/hej小鼠經陰道感染鼠衣原體野生型菌株cmg0和傳代的cmg28,陰道感染cmg28后引起的輸卵管積水病理變化與cmg0感染組相比,明顯降低。因此,傳代的鼠衣原體在體內的致病性與體外細胞感染特性之間存在一個較大的差異。本實驗的目的就是來探尋這種矛盾后面存在的可能機制。方法:體外傳代培養(yǎng)鼠衣原體野生型菌株和質粒缺失株至一定代數,提取基因組dna,通過下一代測序技術對cmg0、cmg28、cmut3g5和cmut3g40進行全基因組測序,比較分析基因組數據;同時動物試驗檢測鼠衣原體的致病性。結果:通過對cmg0和cmg28進行深度基因組分析,cmg28和cmg0的基因組在3個開放讀碼框中存在顯著差異:tc0237(q117e)替代突變(cmg28,100%;cmg0,0%),tc0668(g216*)無義突變(6.4%vs0%)和tc0668(g322r)替代突變(26%vs0%),和多個的tc0412突變多態(tài)性。吸附試驗表明cmg28對細胞的吸附能力增強。cmg28和cmg0經陰道感染c3h/hej小鼠后,二者在小鼠體內有著相似的上行感染能力,而cmg28感染組致輸卵管積水發(fā)生率明顯降低;bio-plex200系統(tǒng)檢測顯示感染cmg28后小鼠輸卵管組織中炎性細胞因子顯著減少,這與輸卵管炎癥細胞浸潤顯著減少相關。tc0237(q117e)是cmg28和cmut3g40共有的突變,提示tc0237(q117e)與鼠衣原體體外吸附能力增強相關。結論:鼠衣原體TC0237(Q117E)突變增強體外吸附能力。鼠衣原體TC0237(Q117E)/TC0668(G322R)突變與鼠衣原體的致病性減弱相關聯(lián)。
[Abstract]:Chlamydia is a strictly parasitic in eukaryotic cells, prokaryotic microorganism has a unique developmental cycle, can cause human and animal diseases, disease complex. Of human pathogenic Chlamydia have chlamydia, Chlamydia pneumoniae and Chlamydia psittaci. Chlamydia infection has become a major public health problem and challenge social problems, has great influence on health and economy. In order to effectively deal with Chlamydia infection to public health challenges, strengthen the basic research of Chlamydia, in-depth study of its pathogenic mechanism, vaccine research garment corpus has become an important research topic in the world Chlamydia research scholars. At present, the rat as a model bacterium Chlamydia has been widely used in human pathogenic and pathogenic mechanism of Chlamydia trachomatis vaccine research. A construction and identification of Chlamydia trachomatis induced mutant plasmid Background and purpose: will play an important role in the Chlamydia trachomatis plasmid carrying strains were pathogenic in comparative studies using plasmid deficient strain and plasmid, genetic function can be observed in addition plasmid, the plasmid deficient strain can also be used as gene engineering bacteria, widely used in molecular biology research; therefore the construction and identification of Chlamydia plasmid deficient strain has important significance methods: the novobiocin treatment in Chlamydia, plaque formation experiment combined with indirect immunofluorescence to detect Pgp3 markers analysis method for screening mutant rat Chlamydia plasmid, further detected by PCR gene plasmid, observe the phenomenon of glycogen accumulation of iodine staining, animal test their pathogenicity. Results: the screening test was carried out in 96 micro holes in 5, by screening the plasmid deficient strain. By PCR analysis indicated that these strains lacking plasmid gene, in addition, in the above The phenomenon of secretion of GlgA protein found no strain infected HeLa cells, no glycogen accumulation. The plasmid deficient strain CMUT3 vaginal infection C3H/HeJ mice no mice induced by hydrosalpinx. Conclusion: the successful construction of rat Chlamydia plasmid deletion strains. Two, in vitro on rat kinuhara body biological characteristics and pathogenic effects of background with the objective: although Chlamydia trachomatis induced in the laboratory has been passed algebra for ten years, the passage selection in the rat Chlamydia outcome has not been reported. Recently, we have to pass Daipei rat trachomatis plasmid mutant cmut3 and wild-type strain, with no auxiliary auxiliary infection and infection "alternate ways were cultured in vitro. Then to observe the infection factors of centrifugal auxiliary dependence when passaged strains infected cells; the passage cmg28 and cmut3g40 in HeLa cells infected with the infection factors of the auxiliary centrifugal Dependent decrease in.C3h/hej mice by Chlamydia trachomatis induced wild type strain cmg0 and cmg28 were vaginal infection, vaginal infection caused by cmg28 after fallopian tube pathology and cmg0 infection group, significantly reduced. Therefore, the passage of rat Chlamydia has a large difference in pathogenicity in vivo and in vitro cell. The infection characteristics the purpose of the experiment is to explore the possible mechanism of this contradiction behind. Methods: in vitro cultured rat Chlamydia wild-type strain and mutant plasmid to a certain algebra, genomic DNA was extracted by next-generation sequencing technology on cmg0, cmg28, cmut3g5 and cmut3g40 for whole genome sequencing, comparative genome analysis and pathogenic data; detection of Chlamydia trachomatis induced animal test. Results: through in-depth analysis of cmg0 genome and cmg28 genome, cmg28 and cmg0 in 3 open reading frames are significant The difference: tc0237 (q117e) mutation (cmg28100%; cmg0,0%), tc0668 (g216*) nonsense mutation (6.4%vs0%) and tc0668 (g322r) mutation (26%vs0%), mutation and multiple tc0412 polymorphism. The adsorption test showed that the adsorption capacity of cmg28 cells enhanced.Cmg28 and cmg0 by c3h/hej after vaginal infection in mice two, have the similar ability of ascending infection in mice, while cmg28 infection group significantly reduced the incidence of hydrosalpinx caused by fallopian; bio-plex200 detection showed cmg28 infection mouse oviduct inflammatory cytokines in tissues was significantly reduced, and the fallopian tube inflammatory cell infiltration was significantly reduced.Tc0237 (q117e) is a common mutation cmg28 and cmut3g40, suggesting that tc0237 (q117e) associated with the adsorption capacity of rat in vitro enhanced. Conclusion: Chlamydia trachomatis TC0237 rats (Q117E) in vitro. TC0237 mutation enhances the adsorption capacity of rat Chlamydia (Q117E) mutation and /TC0668 (G322R) The pathogenicity of Chlamydia is associated with the decrease of Chlamydia.

【學位授予單位】：南華大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R374

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本文編號：1600436