摘要
目的：探讨人羊膜匀浆上清液（hAHS）对脂多糖（LPS）致伤的大鼠肺微血管内皮细胞（RPMVECs）增殖及其分泌促炎因子的影响。方法：用新鲜人羊膜制备hAHS,用考马斯亮蓝法和ELISA法分别测定hAHS中总蛋白和表皮生长因子（EGF）、碱性成纤维细胞生长因子（bFGF）、血管内皮生长因子（VEGF）、白介素-4（IL-4）、IL-10、血管生成素-1（Ang-1）、人β-防御素2（HBD2）的含量。MTT法检测不同体积分数hAHS（0、10%、15%、20%、25%）作用于RPMVECs后细胞增殖活性的变化,确定hAHS促进RPMVECs增殖的最佳浓度。根据共培养条件不同,将RPMVECs随机分为4组：N组（10%FBS＋DMEM/F12）,A组（10%FBS＋DMEM/F12＋15%hAHS）,B组（10%FBS＋DMEM/F12＋LPS）和C组（10%FBS＋DMEM/F12＋15%hAHS＋LPS）。各组细胞在干预后0、12、24、48、72h用MTT法测定吸光度值（A值）,并分别于培养6、8、10、12、24h时用ELISA法测定RPMVECs培养上清液中TNF-α、IL-6、IL-8的含量。结果：hAHS中总蛋白浓度为（725.125±12.625）mg/L,其中EGF、bFGF、VEGF、IL-4、IL-10、Ang-1、HBD2的浓度分别为（504.785±4.665）、（4.426±0.138）、（0.185±0.006）、（25.650±4.104）、（13.733±2.197）、（15.561±0.496）和（4.763±0.714）ng/L。hAHS的体积分数在10%~20%时均具有促进RPMVECs增殖的作用且以15%hAHS培养后第7、9天的增殖率最佳,而25%hAHS在第7、9、11天使RPMVECs增殖率小于0%hAHS组（P〈0.05）。A组48和72h细胞增殖活性较N组显著增加,B组24、48和72h的增殖活性较N组显著降低;C组24、48和72h的增殖活性较B组显著升高（P〈0.05）。ELISA结果显示,B组各时间点的IL-6和TNF-α的含量以及8、10、12和24h的IL-8含量均显著高于N组（P〈0.05）;C组10和12h的IL-6含量,8、10、12和24h的IL-8含量及各时间点的TNF-α含量均显著低于B组（P〈0.05）。结论：hAHS含有促进组织修复、调节免疫反应、降低血管通透性及杀伤致病微生物的多种生长因子、细胞因子,对LPS致伤的RPMVECs增殖具有促进作用,并减少致伤后炎症因子分泌。
Objective：To investigate the protective effect of supernatant of human amnion homogenate（hAHS）on proliferation and expression of proinflammatory mediators by lipopolysaccharide（LPS）induced injured pulmonary microvascular endothelial cells of rats（RPMVECs）.Methods：hAHS was prepared from fresh human amnion.The total protein content and the content of epithelial growth factor（EGF）,basic fibroblast growth factor（bFGF）,vascular endothelial growth factor（VEGF）,interleukin-4（IL-4）,IL-10,angiogenin-1（Ang-1）,humanβ-defensin2（HBD2）of hAHS were determined with Coomassie blue staining and ELISA.The effect of 0,10%,15%,20%,25% hAHS on cell proliferation activity of RPMVECs was respectively determined with MTT assay,in order to determine the optimal concentration of hAHS on promoting RPMVECs proliferation.According to different co-culture conditions,RPMVECs were randomly divided into 4groups：group N（cultured with 10%FBS＋DMEM/F12）,group A（10%FBS＋DMEM/F12＋15%hAHS）,group B（10%FBS＋DMEM/F12＋LPS）,and group C（10%FBS＋DMEM/F12＋15%hAHS＋LPS）.At 0,12,24,48,72 hours after culturing with the corresponding medium of each group,optical density values（Avalues）of each group were determined respectively with MTT assay to determine the proliferation activity,and the contents of IL-6,IL-8,TNF-αlevels in the culture supernates were also determined by ELISA at 6,8,10,12 and 24hours.Results：The total protein concentration of hAHS was（725.125±12.625）mg/L,and levels of EGF,bFGF,VEGF,IL-4,IL-10,Ang-1,HDB2 were respectively（504.785±4.665）ng/L,（4.426±0.138）ng/L,（0.185±0.006）ng/L,（25.650±4.104）ng/L,（13.733±2.197）ng/L,（15.561±0.496）ng/L,（4.763±0.714）ng/L.10%-20% hAHS was shown to promote proliferation of RPMVECs,and 15% hAHS,and the best result was observed on 7and 9days.The proliferation rate of RPMVECs in 25% hAHS group at 7,9and 11 days was lower than those in the 0%hAHS group（P〈0.05）.The proliferation activity（Avalue）of group A was greater than that of group N at 48 and 72hours,and it was lower in group B significantly as compared with that of group N at 24,48 and 72hours（P〈0.05）,while it was significantly higher in group C compared with that of the group B at 24,48 and 72hours（P〈0.05）.ELISA result showed that the contents of IL-6and TNF-αin group B were higher than those of group N（P〈0.05）.IL-8levels of group B at 8,10,12 and 24hours were higher than those of group N（P〈0.05）.In group C,the level of IL-6was significantly lower than that of group B at 10 and 12hours,IL-8level at 8,10,12 and 24hours（P〈0.05）,and TNF-αcontent at each time point（P〈0.05）.Conclusion：hAHS are rich in factors which can promote tissues repair,regulate immune response,reduce vascular permibility,promote hAHS on LPS induced injury of the proliferation of RPMVECs after being injured by LPS,and reduce the secretion of inflammatory cytokines after injury by LPS challenge.
出处
《感染．炎症．修复》
2015年第1期10-16,共7页
Infection Inflammation Repair
基金
国家自然科学基金青年科学基金项目(81301634)
广西科学研究与技术开发计划项目(1140003A-39)
广西自然科学基金(2011GXNSFB018107
2013GXNSFBA019203)
广西卫生厅自筹经费科研课题(Z2014532)
关键词
人羊膜匀浆上清液大鼠肺微血管内皮细胞脂多糖细胞增殖肺损伤
急性
Supernatant of human amnion homogenate Rat Pulmonary microvascular endothelial cells Lipopolisaccharide Cell proliferation Acute
