感染、炎症、修复Issue(1)：10-16,7.DOI:10.3969/j.issn.1672-8521.2015.01.004
人羊膜匀浆上清液对脂多糖致伤的大鼠肺微血管内皮细胞增殖及分泌炎症因子的影响
Effect of supernatant of human amnion homogenate on lipopolysaccharide induced pulmonary microvascular endotheli-al cells injury and their proliferation and expression of proinflammatory factors in rats
摘要
Abstract
Objective:To investigate the protective effect of supernatant of human amnion homogenate (hAHS)on proliferation and expression of proinflammatory mediators by lipopolysaccharide (LPS)induced inj ured 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 or-der to determine the optimal concentration of hAHS on promoting RPMVECs proliferation. According to different co-culture conditions,RPMVECs were randomly divided into 4 groups: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 corre-sponding medium of each group,optical density values (A values)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 su-pernates were also determined by ELISA at 6,8,10,12 and 24 hours. 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 re-spectively(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 prolifer-ation of RPMVECs,and 15% hAHS,and the best result was observed on 7 and 9 days. The proliferation rate of RPMVECs in 25% hAHS group at 7,9 and 11 days was lower than those in the 0%hAHS group (P<0.05).The proliferation activity (A value )of group A was greater than that of group N at 48 and 72 hours,and it was lower in group B significantly as compared with that of group N at 24,48 and 72 hours (P<0.05),while it was signifi-cantly higher in group C compared with that of the group B at 24,48 and 72 hours (P<0.05). ELISA result showed that the contents of IL-6 and TNF-αin group B were higher than those of group N(P<0.05). IL-8 levels of group B at 8,10,12 and 24 hours were higher than those of group N (P<0.05). In group C,the level of IL-6 was significantly lower than that of group B at 10 and 12 hours,IL-8 level at 8,10,12 and 24 hours (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 inj ured by LPS,and reduce the secretion of inflammatory cytokines after inj ury by LPS challenge.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Key words
Supernatant of human amnion homogenate/Rat/Pulmonary microvascular endothelial cells/Lipopolisac-charide/Cell proliferation/Acute lung inj ury引用本文复制引用
陈云鹏,朱富军,龚震宇,辛海明,王磊,童亚林,刘亮,吕璐,莫永亮,詹球,阳齐琼,梁静..人羊膜匀浆上清液对脂多糖致伤的大鼠肺微血管内皮细胞增殖及分泌炎症因子的影响[J].感染、炎症、修复,2015,(1):10-16,7.基金项目
国家自然科学基金青年科学基金项目(81301634) （81301634）
广西科学研究与技术开发计划项目(1140003A-39) （1140003A-39）
广西自然科学基金(2011GXNSFB018107,2013GXNSFBA019203) （2011GXNSFB018107,2013GXNSFBA019203）
广西卫生厅自筹经费科研课题(Z2014532) （Z2014532）
