脂多糖对人肺微血管内皮细胞骨架及TNF-α和IFN-γ表达的影响

Effects of Lipopolysaccharide on the expression of Cytoskeleton,TNF-α and IFN-γ in Human Pulmonary Microvascular Endothelial Cells

目的探讨脂多糖(LPS)直接诱导人肺微血管内皮细胞(HPMVEC)后细胞骨架的改变,肿瘤坏死因子α(TNF-α)、γ干扰素(IFN-γ)mRNA的表达及细胞培养上清液中TNF-α、IFN-γ的表达水平的改变。方法 HPMVEC生长至80%以上融合时,加入LPS以500 ng/mL浓度刺激细胞分别至0、4、6、8 h,经鬼笔环肽染色后采用激光共聚焦显微镜观察HPMVEC细胞骨架的变化。至预定时相点离心收集培养液上清液,利用实时荧光定量RT-PCR法检测TNF-α、IFN-γmRNA的表达,采用ELISA方法测定细胞培养上清液中TNF-α、IFN-γ的表达水平,并进行统计学分析。结果 LPS刺激培养后,8 h组细胞开始出现细胞骨架改变;4 h组TNF-α、IFN-γmRNA表达与0 h组差异无统计学意义(P>0.05),6 h组TNF-α、IFN-γmRNA表达高于0 h组(P<0.05),8 h组TNF-α、IFN-γ表达高于0 h组(P<0.05);4 h组细胞培养上清液TNF-α、IFN-γ的表达水平与0 h组比较差异无统计学意义(P>0.05),6 h组细胞培养上清液TNF-α、IFN-γ的表达水平高于0 h组(P<0.05),8 h组细胞培养上清液TNF-α、IFN-γ的表达水平高于0 h组(P<0.05)。结论细菌致病因子LPS能直接刺激HPM-VEC使细胞结构发生改变,同时影响细胞因子的分泌,使促炎因子TNF-α和IFN-γ分泌升高,参与肺损伤。这可能是LPS发挥其毒性作用诱发急性肺损伤/急性呼吸窘迫综合征的一个重要途径。

Objective To investigate effects of lipopolusaccharides （LPS） on cytoskeleton, and mRNA and protein expression of TNF - α and IFN- γ in human pulmonary microvascnlar endothelial cells （HPMVEC）. Methods LPS stimulation （500 ng/mL） was carried out in HPMVEC, which were cultured till merged by 80% for 0, 4, 6 and 8 hours. The changes of the cytoskeleton were determined by laser scanning confocal microscope （LSCM） with phalloidin stain. The medium supernatant was collected for assessment of both mRNA and protein expression of TNF -α and IFN - γ, by qRT- PCR and ELISA, respectively. Results The human pulmonary microvascular endothelial cells cultured with PLS in medium had the change of cytoskeleton at 8 h group. TNF -α, IFN - γmRNA and protein expression have no signifi- cantly inscrease in 4 h group compared with Oh group （ P 〉 0. 05 ）. TNF-α, IFN - γ mRNA and protein expression signif- icantly inscreased in 6 h group compared with 0 h group （ P 〈 0. 05）. The TNF-α, IFN- γ mRNA expression levels and protein significantly inscreased in the 8 h group than the 0 h group （P 〈 0. 05）. Cytoskeleton modification of HPMEC was observed 8 hours after LPS stimulation. There was no significant difference either in mRNA or protein expression of TNF -α and IFN - γ between 4 h and 0 h groups （P 〉 0. 05）. However, significant increase in both mRNA and protein expression of TNF -α and IFN- γ were observed in 6 h and 8 h groups, when compared with those in 0h group （ P 〈 0. 05 ）. Conclusion Bacterial virulence factor LPS directly leads to the morphological changes of HPMVEC, up - regulates pro - inflammatory cytokines TNF -αand IFN -γ, suggesting that an important pathway, via which LPS, induces ALI/ARDS.