肺泡Ⅱ型上皮细胞A549对TNF-α和LPS的刺激效果研究  

Stimulating effects on alveolar type Ⅱ epithelial A549 cells by TNF-α and LPS

作　　者：黄建华[1,2] 李理[2] 邱娴[2] 张安兵[2] 黄文杰[2] 

机构地区：[1]南方医科大学研究生学院,广州510515 [2]广州军区广州总医院呼吸内科,500010

出　　处：《免疫学杂志》2013年第3期211-215,共5页Immunological Journal

基　　金：国家自然科学基金面上项目(81070003);国家自然科学基金青年基金(81200002);广东省科技计划项目(2011B031800170);广东省科技计划项目(20120318040)

摘　　要：目的研究肺泡Ⅱ型上皮细胞A549对TNF-α和脂多糖(lipopolysaccharides,LPS)刺激的反应效果,探讨急性肺损伤体外细胞模型最佳造模方法。方法体外培养的A549细胞分为正常对照组(normal control,NC组),10 ng/ml TNF-α刺激2 h组,10 ng/ml和100 ng/ml LPS分别刺激2、4、8、16 h组。Western blotting检测胞浆IκB-α含量和IκB-α、NF-κB p65磷酸化水平,qRT-PCR检测细胞TNF-α、IL-1β和IL-6 mRNA转录水平,ELISA检测细胞培养上清IL-1β和IL-6质量浓度。结果 TNF-α刺激导致A549细胞大量凋亡或死亡,而LPS刺激对A549细胞生长形态无明显影响,且与LPS的刺激质量浓度和时间无关。TNF-α刺激可明显增加A549细胞内IκB-α和NF-κB p65磷酸化水平(P<0.05),而LPS刺激对A549细胞内IκB-α和NF-κB p65磷酸化水平无明显影响(P>0.05),且与LPS的刺激质量浓度和时间无关(P>0.05)。TNF-α刺激可显著增加A549细胞内TNF-α、IL-1β、IL-6 mRNA转录水平和培养上清中IL-1β、IL-6质量浓度(P<0.05),而LPS刺激对上述炎症因子的合成和分泌并无显著影响(P>0.05),且与LPS的刺激质量浓度和时间无关(P>0.05)。结论 TNF-α可以有效刺激A549产生过度炎症反应,而LPS不能刺激A549细胞产生炎症损伤,因此建立急性肺损伤体外细胞模型时可以考虑用TNF-α代替LPS刺激。Alveolar type Ⅱ epithelial A549 is ideal cells for studying acute lung injury in vitro, however, there are still more debates about modeling factors. The present study was performed to evaluate the stimulating effects of TNF-α and lipopolysacchride （LPS） on A549 cells and the possible mechanisms. A549 cells were exposured to LPS （10 and 100 ng/ml, respectively） for 2, 4, 8 and 16 h. At same time, 10 ng/ml of TNF-α was used to stimulate A549cells for 2 h. Then Western blotting were performed to investigate the phosphorylation levels of IκB-α and NF-κB p65 in cytoplasm. The transcription levels of TNF-α, IL-1β, and IL-6 in nuclei were detected by Real-timequantitative PCR. The concentrations of L-1β and IL-6 in culture supernatant were detected by ELISA. We found that a large number of A549 cells were death when exposure to TNF-α, however, LPS had no significant effect onthe growth of the A549 cells. Immunoblotting analysis revealed that exposure to TNF-α significantly increased phosphorylation of IκB-α and NF-κB p65 in cytoplasm （P〈 0.05）, however, treatment of A549 cells with LPS didnot increased the phosphorylation of IκB-α and NF-κB p65 in cytoplasm （P〉 0.05）. The transcription intensity of TNF-α, IL-I[5, and IL-6, as well as the secretion of IL-1β and IL-6 in A549 cells were significantly increased byTNF-α stimulating （P 〈 0.05）, however, there had no significant effect on synthesis and secretion of above-mentioned inflammatory cytokines when exposure to LPS （P 〉 0.05）. In conclusion, A549 cells can generateexcessive inflammatory response when exposure to TNF-α, while LPS did not, so we can considered TNF-α, instead of LPS, to stimulate A549 cells when study acute lung injury.

关 键 词：急性肺损伤 A549细胞 TNF-Α 脂多糖 

分 类 号：R563.8[医药卫生—呼吸系统]