【摘要】：背景溶血素(pneumolysin, PLY)是肺炎鏈球菌一個(gè)重要的毒力因子,具有溶細(xì)胞活性、補(bǔ)體激活和誘導(dǎo)細(xì)胞凋亡等多種生物學(xué)功能,但其具體在細(xì)菌感染宿主的哪些環(huán)節(jié)發(fā)揮作用,目前尚不十分清楚。 本研究通過(guò)比較Ply全基因缺陷菌株與野生菌株在定植、侵襲及血中存活能力的差異,確定PLY在細(xì)菌感染過(guò)程中發(fā)揮作用的具體環(huán)節(jié)。結(jié)果顯示,PLY在細(xì)菌損傷肺組織、突破肺部毛細(xì)血管屏障中具有重要作用。而近期研究顯示,在肺炎鏈球菌導(dǎo)致的肺部損傷中,PLY的細(xì)胞凋亡誘導(dǎo)作用較其細(xì)胞毒性作用更強(qiáng),但具體涉及哪些類(lèi)型細(xì)胞尚不清楚。由于肺部巨噬細(xì)胞是這一屏障的重要組成部分,且已有研究顯示PLY可誘導(dǎo)巨噬細(xì)胞的凋亡,提示PLY誘導(dǎo)肺部巨噬細(xì)胞的凋亡可能是其至肺部損傷、幫助細(xì)菌侵襲入血的一個(gè)重要因素。雖有研究顯示這一過(guò)程依賴(lài)于TLR4,但其具體的分子機(jī)制尚不十分清楚。因此,本研究后部分實(shí)驗(yàn)就以小鼠肺泡巨噬細(xì)胞RAW264.7為模型細(xì)胞,探索PLY引起巨噬細(xì)胞凋亡的具體分子機(jī)制,為深入闡明肺炎鏈球菌PLY的致病分子機(jī)制提供有價(jià)值的實(shí)驗(yàn)證據(jù)。 方法采用長(zhǎng)臂同源多聚酶鏈反應(yīng)(Long flanking homologypolymerase chain reaction,LFH-PCR)方法構(gòu)建溶血素缺陷菌株,利用小鼠體內(nèi)實(shí)驗(yàn)研究溶血素對(duì)細(xì)菌毒力的影響;利用純化的PLY蛋白處理RAW264.7細(xì)胞后,通過(guò)倒置顯微鏡的細(xì)胞形態(tài)觀察,MTT增殖實(shí)驗(yàn),DNA Ladder分析,流式細(xì)胞檢測(cè)等鑒定細(xì)胞凋亡;通過(guò)ELISA檢測(cè)Caspase-3、8、9活性,通過(guò)免疫組織化學(xué)分析Bax、Fas、Bcl-2蛋白的表達(dá)情況。 結(jié)果成功構(gòu)建Ply缺陷菌。該缺陷菌株入血時(shí)間(6h)明顯晚于野生菌株(2h),且各時(shí)間點(diǎn)的菌量均顯著低于野生菌株,兩者比較有統(tǒng)計(jì)學(xué)差異(P0.01);缺陷菌株腹腔感染小鼠的中位生存時(shí)間為18天,野生菌株中位生存時(shí)間為3天,兩者比較有統(tǒng)計(jì)學(xué)差異(P0.01)。溶血素處理RAW264.7細(xì)胞后,倒置顯微鏡可見(jiàn)RAW264.7細(xì)胞典型的凋亡形態(tài)學(xué)改變;MTT增殖實(shí)驗(yàn)顯示溶血素對(duì)RAW264.7細(xì)胞有明顯增殖抑制作用,而且呈現(xiàn)出時(shí)間和濃度依賴(lài)性;流式細(xì)胞儀分析結(jié)果顯示,0.5 ug/ml PLY蛋白處理RAW264.7細(xì)胞24h和48h的早期凋亡率分別為7.42%和15.64%,1ug/ml PLY蛋白處理RAW264.7細(xì)胞24h和48h的早期凋亡率分別為43.33%和55.43%(P0.05);瓊脂糖凝膠電泳染色體DNA可見(jiàn)典型的凋亡“梯狀”條帶; 溶血素處理RAW264.7細(xì)胞后,發(fā)現(xiàn)Caspase-3、8、9活性均增高;且凋亡相關(guān)蛋白Bax、Fas表達(dá)增高,Bcl-2表達(dá)降低(P0.05)。 結(jié)論肺炎鏈球菌缺陷溶血素Ply基因后,細(xì)菌的侵襲能力和血中生存能力顯著降低,提示PLY在細(xì)菌損傷肺組織、突破肺部毛細(xì)血管屏障中具有重要作用,其中可能涉及誘導(dǎo)肺泡巨噬細(xì)胞的凋亡。對(duì)RAW264.7細(xì)胞凋亡相關(guān)研究結(jié)果顯示,溶血素可以在體外誘導(dǎo)小鼠巨噬細(xì)胞株RAW264.7細(xì)胞的凋亡,其誘導(dǎo)凋亡的機(jī)制涉及死亡受體/Fas途徑和線(xiàn)粒體途徑的雙重調(diào)控作用。
[Abstract]:Background Hemolysin (pneumolysin, PLY) is an important virulence factor of Streptococcus pneumoniae. It has many biological functions, such as lysocytic activity, complement activation and apoptosis induction. It is not clear yet. The purpose of this study was to compare the difference of colonization, invasion and survival ability between Ply gene deficient strains and wild strains in order to determine the specific links of PLY in the process of bacterial infection. The results showed that ply played an important role in bacterial injury of lung tissue and breakthrough of pulmonary capillary barrier. Recent studies have shown that the apoptosis-inducing effect of ply is stronger than its cytotoxicity in the lung injury induced by Streptococcus pneumoniae, but it is not clear which types of cells are involved. Since pulmonary macrophages are an important part of this barrier, PLY has been shown to induce apoptosis of macrophages, suggesting that apoptosis of pulmonary macrophages induced by PLY may be due to lung injury. An important factor in helping bacteria invade the bloodstream. Although some studies have shown that this process depends on TLR 4, its specific molecular mechanism is not well understood. Therefore, in the later part of this study, the mouse alveolar macrophages (RAW264.7) were used as model cells to explore the specific molecular mechanism of apoptosis induced by PLY, and to provide valuable experimental evidence for further elucidating the pathogenetic molecular mechanism of Streptococcus pneumoniae PLY. Methods the hemolysin deficient strain was constructed by using long arm homologous polymerase chain reaction (Long flanking homologypolymerase chain reactionation (Long flanking homologypolymerase chain LFH-PCR), and the effect of hemolysin on bacterial virulence was studied in vivo. RAW264.7 cells were treated with purified PLY protein. Apoptosis was identified by Ladder analysis and flow cytometry. The activity of Caspase-3 was detected by ELISA and the expression of Bcl-2 protein was analyzed by immunohistochemistry. Results Ply deficient bacteria were successfully constructed. The blood entry time (6h) of the defective strain was significantly later than that of the wild strain (2h), and the amount of bacteria at each time point was significantly lower than that of the wild strain (P0.01), and the median survival time of the mice infected with the defective strain was 18 days. The median survival time of wild strain was 3 days, there was statistical difference between them (P0.01). After hemolysin was treated with RAW264.7 cells, the typical apoptotic morphological changes of RAW264.7 cells were observed under inverted microscope. The results showed that hemolysin could inhibit the proliferation of RAW264.7 cells in a time-and concentration-dependent manner. The results of flow cytometry showed that the early apoptotic rate of RAW264.7 cells treated with 0. 5 ug/ml PLY protein for 24 h and 48 h was 7.42% and 15.64g / ml PLY protein was 43.33% and 55.43% respectively (P0.05), and the early apoptotic rate was 43.33% and 55.43% for RAW264.7 cells treated with PLY protein for 24 h and 48 h, respectively (P0.05). Typical apoptotic "ladder" bands can be seen. After hemolysin treatment of RAW264.7 cells, It was found that the activity of Caspase-3 was increased and the expression of apoptosis-related protein Baxfas was increased and the expression of Bcl-2 was decreased (P0.05). Conclusion Streptococcus pneumoniae is deficient in hemolysin Ply gene, the invasiveness of bacteria and the survival ability in blood are significantly decreased, suggesting that PLY plays an important role in bacterial injury of lung tissue and breakthrough of pulmonary capillary barrier. This may involve inducing apoptosis of alveolar macrophages. Studies on apoptosis of RAW264.7 cells showed that hemolysin could induce apoptosis of mouse macrophage RAW264.7 cells in vitro. The mechanism of apoptosis was involved in the dual regulation of death receptor / FAS pathway and mitochondrial pathway.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R378.1

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