乌司他丁对严重脓毒症血清损伤人肺血管内皮细胞保护作用的实验研究

Protective Effects of Ulinastatin on Human Pulmonary Vascular Endothelial Cells Attacked by Serum from Patients with Septic Shock

目的探讨乌司他丁保护严重脓毒症血清损伤离体人肺血管内皮细胞(PMVECs)的机制。方法离体培养PMVECs,随机分为4组:正常培养组(加入10%胎牛血清,N组)、健康血清组(加入10%健康人血清,H组)、患者血清组(加入10%严重脓毒症患者血清,S组),乌司他丁组(加入1000 U/mL乌司他丁+10%严重脓毒症患者血清,U组);分别于培养0、1、2、4及6 h MTT比色法检测吸光度(OD),观察PMVECs增殖活性变化;ELISA法检测培养上清液的肿瘤坏死因子α(TNF-α)浓度;比较不同时相4组上述指标变化;于培养1 h时,免疫组化观察核转录因子κB(NF-κB)表达情况。结果与N组比较,S组各时间点细胞增殖活性明显下降,U组各时间点细胞增殖活性略下降,S组和U组1、4及6 h细胞增殖活性与N组比较差异均有统计学意义(P<0.05)。与S组比较,U组1、2及6 h细胞增殖活性明显升高(P<0.05)。S组PMVECs多数胞核呈明显阳性表达,U组PMVECs仅少数胞核呈阳性表达,N组及H组PMVECs未见NF-κB表达。与N组比较,S组和U组各时间点TNF-α含量均明显升高(P<0.01),S组与U组各时间点TNF-α含量比较均明显减少(P<0.01)。结论乌司他丁可通过抑制NF-κB活化,减少TNF-α的释放,减轻严重脓毒症血清损伤离体PMVECs程度。

Objective To investigate the protective mechanism of ulinastatin（UTI） in pulmonary microvascular endothelial cells（PMVECs） attacked by serum from the patients with severe sepsis.Methods PMVECs were cultured in vitro and randomly divided into 4 groups,ie.a normal group（culture medium with 10% fetal bovine serum,group N）,a health group（culture medium with 10% healthy human serum,group H）,a patient group（culture medium with 10% human septic shock serum,group S）,and a ulinastatin group（culture medium with 1000 U/mL UTI and 10% human septic shock serum,group U）.The proliferation activity of PMVECs was measured by MTT expressed by optical density（OD）.The concentration of TNF-α in supernatant of culture medium was examined by ELISA at 0,1,2,4,6 hours.The expression of NF-κB was examined by immunohistochemistry at 1 hour.Results Compared with group N,the cell proliferation activity of group S decreased significantly,and the cell proliferation activity of group U decreased slightly at each time point.Compared with group N,the cell proliferation activity of group S and group U at 1,4,6 hours were significant different（P0.05）.Compared with group S,the cell proliferation activity of group U at 1,2,6 hours increased significantly（P0.05）.Obvious positive expression of NF-κB in PMVECs could be seen in group S,a little positive expression in group S,and no expression in group N and group H.Compared with group N,the TNF-α levels of group S and group U increased significantly at each time point with significant differences（P0.01）.Compared with group S,the TNF-α levels were significantly reduced at each time point in group U（P0.01）.Conclusions UTI can reduce the release of TNF-α by inhibiting NF-κB activation,thus reduce PMVECs injury attacked by serum from severe sepsis patients.