自噬体在肺炎链球菌感染过程中的保护作用机制探讨

The effect of autophagy on Streptococcus pneumoniae infections of lung epithelial cells and its mechanism

目的:检测微管相关蛋白轻链-3(LC3)在肺泡Ⅱ型上皮细胞A549上的表达情况,及3-甲基腺嘌呤(3MA)刺激后对其表达的影响,为研究自噬体在抵抗肺炎链球菌感染过程中的保护作用奠定基础。方法:体外培养肺泡Ⅱ型上皮细胞A549,在肺炎链球菌感染A549细胞12h时用倒置显微镜拍照并提取RNA,采用RT-PCR的方法检测LC3mRNA的表达情况。同时检测对照组、3MA组、Spn组和3MA+Spn组上清液乳酸脱氢酶(LDH)的OD值。结果:RT-PCR检测LC3mRNA有明显表达,3MA可以抑制LC3的表达。LDH检测显示Spn组和3MA+Spn组上清液LDH的OD值数据两两比较差异有统计学意义(P<0.05)。结论:LC3在肺泡Ⅱ型上皮细胞中显著表达,3MA可以抑制细胞的自噬作用。加3MA后,肺炎链球菌感染肺泡Ⅱ型上皮细胞坏死增加,提示自噬体在抵抗肺炎链球菌的感染过程中起一定的保护作用。

Objective To detect the expression of LC3 in human alveolar type Ⅱ epithelial cells A549 and the effect of Streptococcus pneumoniae and 3MA on it, lay the foundation for studying on autophagosome resistance in the process of Streptococcus pneumoniae infection. Methods Human pulmonary type Ⅱ epithelial cells cultured in vitro and stimulated with Streptococcus pneumoniae. Extract the RNA of A549 cells on 12 h and detect LC3 mRNA expression by RT-PCR. The necrosis of control group, 3-MA group, Spn group and 3-MA ＋ Spn group vcere detected by the necrosis kit after 12h by Non-Radioactive cytotoxieity assay respectively. Results The expression of LC3 mRNA stimulated of 3MA deteced by RT-PCR was different. The necrosis test showed the blank group and 3-MA group was not significantly different （P 〉 0.05）, Spn group and 3-MA ＋ Spn group significantly different （P 〈 0.05）. Conclusions The expression of LC3 mRNA in alveolar type Ⅱ epithelial cells stimulated with Streptococcus pneumoniae was significantly different. The necrosis number of alveolar type Ⅱepithelial cells which added 3MA increased, suggesting that autophagosome is a defense mechanism in infected lung epithelial cells.