冷藏对新鲜冰冻血浆诱导内皮细胞迁移的影响及其分子机制研究  

Effect of Fresh Frozen Plasma on Endothelial Cell Migration Diminishes During Storage and Its Molecular Mechanisms

作　　者：张春芳[1] 谭潭[1,2] 李建国[1] 马虹英[1] 陈主初[1] 段朝军[1,3] 

机构地区：[1]中南大学湘雅医院卫生部肿瘤蛋白质组学重点实验室,长沙410008 [2]郴州市第一人民医院检验科,郴州423000 [3]中南大学湘雅医院医学实验研究中心,长沙410008

出　　处：《生物化学与生物物理进展》2011年第6期528-535,共8页Progress In Biochemistry and Biophysics

基　　金：国家重点基础研究发展计划(973)(2001CB510207);国家自然科学基金(30670990;30871189)资助项目~~

摘　　要：探讨冷藏是否导致新鲜冰冻血浆(fresh frozen plasma,FFP)中的转化生长因子-β(transform growth factor-β,TGF-β)发生变化并降低其对内皮细胞迁移的诱导能力及其分子机制.为证实冷藏可能导致FFP中TGF-β的水平升高进而影响其功能,首先采用ELISA法分析当天解冻FFP(FFP Day 0)和解冻后4℃冷藏1～5天FFP中TGF-β含量,迁移实验及Western blot分析比较FFP(Day 0)和FFP(Day 5)处理人肺微血管内皮细胞(human pulmonary microvascular endothelial cells,HPMECs)后的细胞迁移率及TGF-β信号通路Smad2和Smad3磷酸化,进一步采用ALK5-siRNA和ALK5特异性抑制剂阻断细胞TGF-βⅠ型受体ALK5的活性,分析下调TGF-β/Smad2/3信号传导后的内皮细胞迁移率.结果显示:随着冷藏时间的延长,FFP中TGF-β1水平逐渐增加,其增加率为244.31 ng/(L.d)(P<0.05);与FFP(Day 0)相比,FFP(Day 5)诱导内皮细胞HPMECs的TGF-β信号通路Smad2/3磷酸化显著增加(P<0.05);不管是FFP(Day 0)还是FFP(Day 5)体外均能诱导内皮细胞迁移,与FFP(Day 0)相比,FFP(Day 5)诱导细胞迁移能力显著降低(P<0.05);阻断TGF-βⅠ型受体ALK5的活性、下调Smad 3信号传导可恢复FFP(Day5)诱导细胞迁移能力.上述结果表明,FFP能显著诱导内皮细胞迁移,4℃冷藏增加FFP中TGF-β1水平并增加内皮细胞TGF-β1信号传导,进而降低FFP诱导内皮细胞迁移.提示,FFP复苏疗效的提高可能与增加内皮细胞迁移有关,冷藏导致内皮细胞迁移降低进而降低FFP复苏疗效.To determine whether TGF-βs and the effect on endothelial cell migration were altered during fresh frozen plasma （FFP） refrigeration, ELISA assay was carried out to quantify TGF-βs protein levels in FFP stored at 4 ℃ for up to 5 days. Human pulmonary microvascular endothelial cells （HPMECs） were treated with various concentrations of Day 0 and Day 5 FFPs or 10% Lactated Ringer＇s （LR） and were subjected to migration assay or Western blot analysis. ALK5 siRNA or ALK5 inhibitor were used to block FFP-induced Smad3 signaling in EC cells. It was found that TGF-β1 protein levels increased in a time-dependent fashion at a rate of 244.31 ng/（L ·d） （P 〈 0.05） and greater activation of its downstream mediators Smad2/3 during storage of FFP （P 〈 0.05）. Both Day 0 FFP and Day 5 FFP stimulated EC migration in vitro; however, the effect of Day 5 FFP was significantly reduced. Inhibition of TGF-β type I receptor blocked FFP-induced Smad3 signaling in EC cells and restored the effectiveness of Day 5 FFP on EC migration to a comparable level as Day 0 FFP. These data suggest that the increased TGF-β levels during FFP storage contributes to the deterioration of stored FFP＇s effects on EC migration. A novel molecular mechanism contributing to the reduced efficacy of stored FFP was identified.

关 键 词：冷藏 新鲜冰冻血浆 内皮细胞 迁移 TGF—β/ALK5/Smad2/3 

分 类 号：R459.9[医药卫生—治疗学]