肺炎链球菌溶血素对细菌毒力的影响及诱导RAW264.7细胞凋亡的研究

发布时间：2018-07-26 20:51

【摘要】：背景溶血素(pneumolysin, PLY)是肺炎链球菌一个重要的毒力因子,具有溶细胞活性、补体激活和诱导细胞凋亡等多种生物学功能,但其具体在细菌感染宿主的哪些环节发挥作用,目前尚不十分清楚。本研究通过比较Ply全基因缺陷菌株与野生菌株在定植、侵袭及血中存活能力的差异,确定PLY在细菌感染过程中发挥作用的具体环节。结果显示,PLY在细菌损伤肺组织、突破肺部毛细血管屏障中具有重要作用。而近期研究显示,在肺炎链球菌导致的肺部损伤中,PLY的细胞凋亡诱导作用较其细胞毒性作用更强,但具体涉及哪些类型细胞尚不清楚。由于肺部巨噬细胞是这一屏障的重要组成部分,且已有研究显示PLY可诱导巨噬细胞的凋亡,提示PLY诱导肺部巨噬细胞的凋亡可能是其至肺部损伤、帮助细菌侵袭入血的一个重要因素。虽有研究显示这一过程依赖于TLR4,但其具体的分子机制尚不十分清楚。因此,本研究后部分实验就以小鼠肺泡巨噬细胞RAW264.7为模型细胞,探索PLY引起巨噬细胞凋亡的具体分子机制,为深入阐明肺炎链球菌PLY的致病分子机制提供有价值的实验证据。方法采用长臂同源多聚酶链反应(Long flanking homologypolymerase chain reaction,LFH-PCR)方法构建溶血素缺陷菌株,利用小鼠体内实验研究溶血素对细菌毒力的影响;利用纯化的PLY蛋白处理RAW264.7细胞后,通过倒置显微镜的细胞形态观察,MTT增殖实验,DNA Ladder分析,流式细胞检测等鉴定细胞凋亡;通过ELISA检测Caspase-3、8、9活性,通过免疫组织化学分析Bax、Fas、Bcl-2蛋白的表达情况。结果成功构建Ply缺陷菌。该缺陷菌株入血时间(6h)明显晚于野生菌株(2h),且各时间点的菌量均显著低于野生菌株,两者比较有统计学差异(P0.01);缺陷菌株腹腔感染小鼠的中位生存时间为18天,野生菌株中位生存时间为3天,两者比较有统计学差异(P0.01)。溶血素处理RAW264.7细胞后,倒置显微镜可见RAW264.7细胞典型的凋亡形态学改变;MTT增殖实验显示溶血素对RAW264.7细胞有明显增殖抑制作用,而且呈现出时间和浓度依赖性;流式细胞仪分析结果显示,0.5 ug/ml PLY蛋白处理RAW264.7细胞24h和48h的早期凋亡率分别为7.42%和15.64%,1ug/ml PLY蛋白处理RAW264.7细胞24h和48h的早期凋亡率分别为43.33%和55.43%(P0.05);琼脂糖凝胶电泳染色体DNA可见典型的凋亡“梯状”条带; 溶血素处理RAW264.7细胞后,发现Caspase-3、8、9活性均增高;且凋亡相关蛋白Bax、Fas表达增高,Bcl-2表达降低(P0.05)。结论肺炎链球菌缺陷溶血素Ply基因后,细菌的侵袭能力和血中生存能力显著降低,提示PLY在细菌损伤肺组织、突破肺部毛细血管屏障中具有重要作用,其中可能涉及诱导肺泡巨噬细胞的凋亡。对RAW264.7细胞凋亡相关研究结果显示,溶血素可以在体外诱导小鼠巨噬细胞株RAW264.7细胞的凋亡,其诱导凋亡的机制涉及死亡受体/Fas途径和线粒体途径的双重调控作用。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378.1

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