热打击联合脂多糖刺激对人肠上皮细胞SW480释放细胞因子的影响

Effects of lipopolysaccharide combined with heat stress on the expression and secretion of cytokines released from human intestinal epithelial cells SW480

目的:观察脂多糖(LPS)与热打击分别单独作用与联合作用对人肠上皮细胞SW480释放细胞因子的影响。方法:于37℃、5%CO_2标准培养箱中培养人肠上皮细胞株SW480。实验分4组:空白对照组直接收集细胞上清;LPS单独刺激组(LPS组)分别使用0、0.01、0.1、1和10μg/ml的LPS刺激SW480细胞,24 h后收集细胞上清;热打击组将SW480细胞进行热打击(42℃,1 h)后在标准孵箱分别培养1 h和24 h后收集细胞上清;LPS+热打击组将SW480细胞42℃热打击1 h后给予上述浓度的LPS刺激,再重新置于标准孵箱中培养,24 h后收集细胞上清。各组细胞上清用LiquiChip液相蛋白芯片系统分别检测粒细胞-巨噬细胞集落刺激因子(GMCSF)、γ干扰素(IFN-γ)、白细胞介素-1β(IL-1β)、IL-2、IL-4、IL-6、IL-8、IL-10、IL-12、肿瘤坏死因子-α(TNF-α)等10种细胞因子/趋化因子的表达分泌水平。结果:单独LPS刺激时呈剂量依赖性诱导SW480表达分泌IL-8,对其余9种细胞因子/趋化因子的表达分泌水平影响不明显。单纯热打击仅诱导SW480细胞表达分泌IL-8的水平升高,且与空白对照组比较,仅热打击24 h后IL-8的水平显著增加(P<0.01)。热打击联合不同浓度的LPS刺激SW480细胞,LPS浓度低时并不影响SW480表达分泌IL-8的水平(P>0.05),而高浓度LPS可以显著减少其表达分泌水平(P<0.05)。结论:人肠上皮细胞SW480作为脏器实质细胞,对炎性刺激仅能产生单一种类的细胞因子,其中LPS单独刺激和一定强度的热打击均可上调SW480分泌IL-8;但高浓度LPS在热环境下可抑制SW480细胞的IL-8表达分泌水平。

Objective： To study the effects of lipopolysaccharide （LPS） and heat stress alone or in combination on the expression and secretion of cytokines released from human intestinal epithelial cell line （SW480）. Methods： SW480 cells were cultured in standard condition of 37 ℃ in a 5% carbon dioxide incubator and randomly divided into 4 groups. The supernatant of SW480 culture in blank control group was collected directly. To LPS group different concentration of LPS （0, 0.01, 0. 1, 1, 10 μg/ml） was added, and the supernatant of the culture after 24 hours of stimulation was collected. In heat stress （HS） group the-culture was exposed to 42 ℃ for 1 hour, and it was cultured again in the incubator, and the supernatant was collected after 1 hour and 24 hours, respectively. In LPS ＋ HS group cultured cells were exposed to heat exposure, followed by the addition of LPS in above men- tioned concentration of LPS, and the supernatant was collected after 24 hours of combined stimulation. The levels of granulocyte/macrophage colony stimulating factor （GM-CSF）, interferon-） （IFN-7）, tumor necrosis factor-α （TNF-α）, and interleukin-1β （IL-1β）, IL-2, IL-4, IL-6, IL-8, IL-10, and IL-12 levels in the supernatant were determined by LiquiChip system. Results： In LPS group, the expression and secretion of IL-8 from SW480 was in- creased in a dose-dependent manner, but no changes in level of other cytokines was observed. In HS group, heat exposure only induced elevated expression and secretion of IL-8, with no effect on other cytokines. Compared with blank control group, only the expression and level of IL-8 in the supernatant of HS group were significantly in- creased 24 hours after heat stress （P〈0.01）. In HS-bLPS group, the expression and level of IL-8 were not changed after heat stress combined with low-level LPS stimulation （P^0. 05）, but it decreased significantly after heat stress com- bined with high-level LPS stimulation （P〈0. 05）. Conclusions： Inflammatory stimulation can only facilitate human intes- tinal epithelial cells （SW480） to secrete and express single cytokin Either the LPS stimulation or heat exposure at cer- tain intensity can up-regulate the expression and secretion of IL-8. However, the cells are exposed to combined high-level LPS stimulation and heat stress, the expression and secretion of IL-8 is significantly decreased.