Objective To investigate effects of lipopolusaccharides ( LPS ) on cytoskeleton, and mRNA and protein expression of TNF - α and IFN - 7 in human pulmonary microvascular 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 - y mRNA and protein expression have no significantly inscrease in 4 h group compared with Oh group ( P > 0. 05 ). TNF - α, IFN - y mRNA and protein expression significantly inscreased in 6 h group compared with 0 h group ( P < 0. 05 ). The TNF - α, IFN - y 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 - y between 4 h and 0 h groups ( P >0. 05 ). However, significant increase in both mRNA and protein expression of TNF - α and IFN - y were observed in 6 h and 8 h groups, when compared with those in 0 h group ( P <0. 05 ). Conclusion Bacterial virulence factor LPS directly leads to the morphological changes of HPMVEC, up - regulates pro -inflammatory cytokines TNF - α and IFN - γ, suggesting that an important pathway, via which LPS, induces ALI/ARDS.%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-γ mRNA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-γ mRNA表达与0 h组差异无统计学意义(P>0.05),6 h组TNF-α、IFN-γ mRNA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HPMVEC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.
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