微生物學(xué)通報(bào) MAY 20,2010,37(5):776～782
自噬對(duì)鼠傷寒沙門(mén)菌所致的巨噬細(xì)胞凋亡的影響
吳淑燕 李瓊 儲(chǔ)元元 李嫄淵 黃瑞* 秦正紅*
(蘇州大學(xué)醫(yī)學(xué)部基礎(chǔ)醫(yī)學(xué)與生物科學(xué)學(xué)院 江蘇 蘇州 215123)
摘 要: 為探討鼠傷寒沙門(mén)菌與巨噬細(xì)胞共作用時(shí)細(xì)胞自噬對(duì)凋亡的影響, 用加入自噬誘導(dǎo)劑雷帕霉素(Rapamycin, RAPA)和未加RAPA的RPMI 1640過(guò)夜培養(yǎng)小鼠腹腔巨噬細(xì)胞J774A.1, 以攜帶一分子量為100 kb毒力質(zhì)粒的鼠傷寒沙門(mén)菌標(biāo)準(zhǔn)毒株SR-11為受試菌。首先測(cè)定RAPA對(duì)菌量及細(xì)胞活性的影響, 然后建立細(xì)胞感染模型, 在細(xì)菌與細(xì)胞共作用后動(dòng)態(tài)觀察24 h, 不同時(shí)間點(diǎn)檢測(cè)細(xì)胞超微結(jié)構(gòu)變化,、自噬泡的形成,、Beclin-1和Bcl-2的表達(dá)、細(xì)胞存活率和胞內(nèi)活菌計(jì)數(shù)以及細(xì)胞凋亡情況,。結(jié)果顯示, RAPA單獨(dú)作用于細(xì)菌或細(xì)胞時(shí)菌量及細(xì)胞活性均無(wú)變化; 而對(duì)細(xì)胞感染模型而言, RAPA作用與否細(xì)胞內(nèi)的細(xì)菌數(shù)及細(xì)胞存活率均有顯著改變, RAPA可明顯降低細(xì)胞內(nèi)活菌數(shù)及其所致的巨噬細(xì)胞凋亡率(P < 0.05); RAPA干預(yù)組在細(xì)菌與細(xì)胞共作用早期, 部分細(xì)菌可被雙層膜包裹形成自噬泡, 細(xì)胞超微結(jié)構(gòu)正常; Beclin-1的表達(dá)量增加, 而B(niǎo)cl-2的表達(dá)量降低; 后期細(xì)胞破壞程度明顯輕于未用RAPA組,。以上結(jié)果提示, 通過(guò)調(diào)控細(xì)胞自噬水平以減輕宿主細(xì)胞凋亡, 可作為防治某些感染性疾病的新途徑。
關(guān)鍵詞: 鼠傷寒沙門(mén)菌, 巨噬細(xì)胞, 自噬, 凋亡
The Influence of Autophagy on Apoptosis of Macrophage Induced by Salmonella typhimurium
WU Shu-Yan LI Qiong CHU Yuan-Yuan LI Yuan-Yuan HUANG Rui* QIN Zheng-Hong*
(Medical College of Soochow University, Suzhou, Jiangsu 215123, China)
Abstract: In the present study, the influence of autophagy on apoptosis of macrophage induced by Salmonella typhimurium was investigated. Murine macrophage-like cell line J774A.1 was cultivated with RPMI 1640 containing autophagy inducer rapamycin (RAPA) or not overnight, the standard Sal-monella typhimurium virulent strain SR-11 was used as the test bacterium. First, the influence of RAPA on bacteria growth and J774A.1 cells survival were detected, then the incubation of J774A.1 and SR-11 were dynamically tracked 24 h. Cell ultrastructure, the formation of autophagic vesicles, Beclin-1 and Bcl-2 expression, cell survival rate, the number of intracellular viable bacteria and cell apoptosis were detected at different time. The results showed that RAPA didn't affect bacteria growth and the survival of J774A.1 cells. However, in cellular infectious model, RAPA could significantly reduce the number of intracellular viable bacteria and the rate of macrophage apoptosis, thereby increasing cell survival (P < 0.05). Some bacteria was wrapped in the autophagic vacuoles of J774A.1 cells during inchoate infec-tious stage, and cellular ultrastructure was normal after RAPA treatment. Moreover, the expression of Beclin-1 was increased while the expression of Bcl-2 was declined, and cells damage were significantly lighter in the late stage. All these results suggested that the regulation of cellular autophagy to lower the level of host cells apoptosis may be used as new ways of the prevention and control for some infectious diseases.
Keywords: Salmonella typhimurium, Macrophage, Autophagy, Apoptosis
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