血小板微粒及其携带的sCD40L在输血相关急性肺损伤中作用的初步研究
发布时间：2018-10-12 06:44
【摘要】：血小板输注在临床输血中具有重要的意义。然而血小板储存过程中可能发生不同程度的活化,分泌血小板微粒(platelet-derived microparticles, PMPs)和可溶性CD40配体(soluble CD40ligand, sCD40L, CD154)等一系列具有重要的生物学功能的促炎因子。在其刺激下,多形核中性粒细胞(polymorphonuclear neutrophil, PMNs)可快速移位至肺毛细血管部位,经引发活化后产生呼吸爆发,释放大量的超氧阴离子和活性氧,导致组织损伤,诱发输血相关急性肺损伤(TRALI)的发生。 本文主要研究血小板储存过程中,储存血小板血浆(以下简称血浆)及PMPs引发PMNs呼吸爆发,介导人肺微血管内皮细胞(HMVECs)损伤的作用。重点讨论储存血小板制品中PMPs的性质和生物学功能以及SCD40L的作用。 本研究取储存第1天、第3天和第5天的单采血小板(A-PLTs)和白膜法浓缩血小板(BC-PLTs)样品低速离心分离获得血浆,取血浆超速离心获得PMPs。 ELISA检测血浆及PMPs中sCD40L的含量；抗体抑制实验评价血浆中sCD40L的作用。免疫电子显微镜观察PMPs形态及sCD40L的表达。Western Blotting检测携带的CD40L蛋白分子的存在形式。以绝对细胞计数微球通过流式细胞术检测血PMPs浆中数量。用活性氧特异性荧光探针二氢若丹明123(DHR)流式细胞术PMPs检测呼吸爆发。建立(?)PMNs两次打击细胞模型,评价TRALI对PMPs介导的PMNs损伤的作用。HMVECs结果显示,在血小板1-5天储存过程中, 血浆中BC-PLTs含量逐渐递sCD40L增,第3天达到峰值(P0.05)；而血浆中A-PLTs含量第3天即显著增加sCD40L(P0.01),第5天达到峰值。随血小板储存时间延长,血浆引发活化的fML呼吸爆发的程度亦逐渐增强,储存第3天的血浆比第1天的血浆对PMNs的引发活性显著提高(P0.05)。A-PLTs血浆对PMNs的引发活性高于BC-PLTs血PMNs浆。用小鼠抗人CD154抗体特异性去除血浆中的A-PLTs或用0.1μm滤器滤除后,可部分抑制血浆引发sCD40L活化的PMPs呼吸爆发。fMLP电子显微镜下观察PMNs呈1μm的囊泡结构,表面携带CD40L且呈不均匀分 PMPs布。Western Blotting结果显示PMPs所携带的CD40L分子量处于26-34kd之间。随A-PLTs储存时间的延长,血浆中PMPs数量及表面携带的sCD40L的量增加,PMPs引发呼吸爆发的程度增强,储存第3天比储存第1天的血浆中分离获得的PMPs引发PMN呼吸爆发的活性显著增强(p0.05)。TRALI两次打击细胞模型实验显示,储存血小板制品中分离的PMPs可引发LPS活化的PMNs呼吸爆发,导致HMVECs的损伤。 综上所述,目前血库血小板储存条件(22℃,5天)下,血浆中PMPs数量和sCD40L含量升高。PMPs表面携带的sCD40L可能是在PMPs引发PMNs呼吸爆发,进而诱导HMVEC损伤的效应分子之一。由此证明,输注血库储存的血小板制品可能对TRALI的发生具有一定的影响。进一步研究PMP及所携带的sCD40L引发PMN呼吸爆发的机制,将为阐明TRALI的发病机制提供相关的实验依据。
[Abstract]:Platelet transfusion plays an important role in clinical blood transfusion. However, platelet activation may occur in varying degrees during platelet storage, and platelet particulates (platelet-derived microparticles, PMPs) and soluble CD40 ligand (soluble CD40ligand, sCD40L, CD154) may be secreted as a series of important biological pro-inflammatory factors. Under its stimulation, polymorphonuclear neutrophils (polymorphonuclear neutrophil, PMNs) could be rapidly translocated to the pulmonary capillaries. After activation, respiratory burst was produced, and a large number of superoxide anion and reactive oxygen species were released, which resulted in tissue damage. Induction of transfusion-associated acute lung injury (TRALI). The purpose of this study was to investigate the role of stored platelet plasma (plasma) and PMPs in inducing respiratory burst of PMNs and mediating (HMVECs) damage of human pulmonary microvascular endothelial cells during platelet storage. The properties and biological functions of PMPs and the role of SCD40L in platelet storage were discussed. In this study, plasma was isolated from single platelets (A-PLTs) and white membrane concentrated platelets (BC-PLTs) samples on day 1, day 3 and day 5 after storage. Plasma was obtained by ultracentrifugation, and PMPs. was obtained by ultracentrifugation. The levels of sCD40L in plasma and PMPs were detected by ELISA, and the effect of sCD40L in plasma was evaluated by antibody inhibition test. The morphology of PMPs and the expression of sCD40L were observed by immunoelectron microscopy. Western Blotting was used to detect the existence of CD40L protein. Absolute cell count microspheres were used to detect the number of PMPs plasma by flow cytometry. Respiratory burst was detected by reactive oxygen species specific fluorescence probe dihydrorhodamine 123 (DHR) flow cytometry PMPs. The effect of TRALI on PMNs damage mediated by PMPs was evaluated by establishing two cell attack models of PMNs. HMVECs results showed that during the 1-5 days of platelet storage, the content of BC-PLTs in plasma increased gradually and reached the peak value on the third day (P0.05). On the 3rd day, the content of A-PLTs in plasma increased sCD40L significantly (P0.01), and reached the peak on the 5th day. With the prolongation of platelet storage time, the degree of activated fML respiratory burst induced by plasma was gradually increased, and the initiation activity of A-PLTs plasma to PMNs was significantly higher than that of BC-PLTs plasma PMNs plasma on the third day of storage compared with that on the first day (P0.05). The specific removal of A-PLTs from plasma with mouse anti-human CD154 antibody or filtering with 0.1 渭 m filter could partially inhibit the PMPs respiratory burst induced by sCD40L activation in plasma. The vesicle structure of PMNs was observed by fMLP electron microscope. The results of. Western Blotting showed that the molecular weight of CD40L carried by PMPs was in the range of 26-34kd. With the prolongation of A-PLTs storage time, the number of PMPs in plasma and the amount of sCD40L carried on the surface increased, and the degree of respiratory burst induced by PMPs increased. The activity of PMN respiratory burst induced by PMPs isolated from plasma on the third day of storage was significantly higher than that on the first day of storage. (p0.05). TRALI two cell model experiments showed that isolated PMPs from stored platelet products could induce PMNs respiratory burst activated by LPS. Damage to HMVECs. In conclusion, the amount of PMPs and the content of sCD40L in plasma increased under the current condition of platelet storage (22 鈩，
本文编号：2265166
[Abstract]:Platelet transfusion plays an important role in clinical blood transfusion. However, platelet activation may occur in varying degrees during platelet storage, and platelet particulates (platelet-derived microparticles, PMPs) and soluble CD40 ligand (soluble CD40ligand, sCD40L, CD154) may be secreted as a series of important biological pro-inflammatory factors. Under its stimulation, polymorphonuclear neutrophils (polymorphonuclear neutrophil, PMNs) could be rapidly translocated to the pulmonary capillaries. After activation, respiratory burst was produced, and a large number of superoxide anion and reactive oxygen species were released, which resulted in tissue damage. Induction of transfusion-associated acute lung injury (TRALI). The purpose of this study was to investigate the role of stored platelet plasma (plasma) and PMPs in inducing respiratory burst of PMNs and mediating (HMVECs) damage of human pulmonary microvascular endothelial cells during platelet storage. The properties and biological functions of PMPs and the role of SCD40L in platelet storage were discussed. In this study, plasma was isolated from single platelets (A-PLTs) and white membrane concentrated platelets (BC-PLTs) samples on day 1, day 3 and day 5 after storage. Plasma was obtained by ultracentrifugation, and PMPs. was obtained by ultracentrifugation. The levels of sCD40L in plasma and PMPs were detected by ELISA, and the effect of sCD40L in plasma was evaluated by antibody inhibition test. The morphology of PMPs and the expression of sCD40L were observed by immunoelectron microscopy. Western Blotting was used to detect the existence of CD40L protein. Absolute cell count microspheres were used to detect the number of PMPs plasma by flow cytometry. Respiratory burst was detected by reactive oxygen species specific fluorescence probe dihydrorhodamine 123 (DHR) flow cytometry PMPs. The effect of TRALI on PMNs damage mediated by PMPs was evaluated by establishing two cell attack models of PMNs. HMVECs results showed that during the 1-5 days of platelet storage, the content of BC-PLTs in plasma increased gradually and reached the peak value on the third day (P0.05). On the 3rd day, the content of A-PLTs in plasma increased sCD40L significantly (P0.01), and reached the peak on the 5th day. With the prolongation of platelet storage time, the degree of activated fML respiratory burst induced by plasma was gradually increased, and the initiation activity of A-PLTs plasma to PMNs was significantly higher than that of BC-PLTs plasma PMNs plasma on the third day of storage compared with that on the first day (P0.05). The specific removal of A-PLTs from plasma with mouse anti-human CD154 antibody or filtering with 0.1 渭 m filter could partially inhibit the PMPs respiratory burst induced by sCD40L activation in plasma. The vesicle structure of PMNs was observed by fMLP electron microscope. The results of. Western Blotting showed that the molecular weight of CD40L carried by PMPs was in the range of 26-34kd. With the prolongation of A-PLTs storage time, the number of PMPs in plasma and the amount of sCD40L carried on the surface increased, and the degree of respiratory burst induced by PMPs increased. The activity of PMN respiratory burst induced by PMPs isolated from plasma on the third day of storage was significantly higher than that on the first day of storage. (p0.05). TRALI two cell model experiments showed that isolated PMPs from stored platelet products could induce PMNs respiratory burst activated by LPS. Damage to HMVECs. In conclusion, the amount of PMPs and the content of sCD40L in plasma increased under the current condition of platelet storage (22 鈩，
本文编号：2265166
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