血小板微粒及其攜帶的sCD40L在輸血相關急性肺損傷中作用的初步研究
發(fā)布時間：2018-10-12 06:44
【摘要】：血小板輸注在臨床輸血中具有重要的意義。然而血小板儲存過程中可能發(fā)生不同程度的活化,分泌血小板微粒(platelet-derived microparticles, PMPs)和可溶性CD40配體(soluble CD40ligand, sCD40L, CD154)等一系列具有重要的生物學功能的促炎因子。在其刺激下,多形核中性粒細胞(polymorphonuclear neutrophil, PMNs)可快速移位至肺毛細血管部位,經引發(fā)活化后產生呼吸爆發(fā),釋放大量的超氧陰離子和活性氧,導致組織損傷,誘發(fā)輸血相關急性肺損傷(TRALI)的發(fā)生。 本文主要研究血小板儲存過程中,儲存血小板血漿(以下簡稱血漿)及PMPs引發(fā)PMNs呼吸爆發(fā),介導人肺微血管內皮細胞(HMVECs)損傷的作用。重點討論儲存血小板制品中PMPs的性質和生物學功能以及SCD40L的作用。 本研究取儲存第1天、第3天和第5天的單采血小板(A-PLTs)和白膜法濃縮血小板(BC-PLTs)樣品低速離心分離獲得血漿,取血漿超速離心獲得PMPs。 ELISA檢測血漿及PMPs中sCD40L的含量；抗體抑制實驗評價血漿中sCD40L的作用。免疫電子顯微鏡觀察PMPs形態(tài)及sCD40L的表達。Western Blotting檢測攜帶的CD40L蛋白分子的存在形式。以絕對細胞計數微球通過流式細胞術檢測血PMPs漿中數量。用活性氧特異性熒光探針二氫若丹明123(DHR)流式細胞術PMPs檢測呼吸爆發(fā)。建立(?)PMNs兩次打擊細胞模型,評價TRALI對PMPs介導的PMNs損傷的作用。HMVECs結果顯示,在血小板1-5天儲存過程中, 血漿中BC-PLTs含量逐漸遞sCD40L增,第3天達到峰值(P0.05)；而血漿中A-PLTs含量第3天即顯著增加sCD40L(P0.01),第5天達到峰值。隨血小板儲存時間延長,血漿引發(fā)活化的fML呼吸爆發(fā)的程度亦逐漸增強,儲存第3天的血漿比第1天的血漿對PMNs的引發(fā)活性顯著提高(P0.05)。A-PLTs血漿對PMNs的引發(fā)活性高于BC-PLTs血PMNs漿。用小鼠抗人CD154抗體特異性去除血漿中的A-PLTs或用0.1μm濾器濾除后,可部分抑制血漿引發(fā)sCD40L活化的PMPs呼吸爆發(fā)。fMLP電子顯微鏡下觀察PMNs呈1μm的囊泡結構,表面攜帶CD40L且呈不均勻分 PMPs布。Western Blotting結果顯示PMPs所攜帶的CD40L分子量處于26-34kd之間。隨A-PLTs儲存時間的延長,血漿中PMPs數量及表面攜帶的sCD40L的量增加,PMPs引發(fā)呼吸爆發(fā)的程度增強,儲存第3天比儲存第1天的血漿中分離獲得的PMPs引發(fā)PMN呼吸爆發(fā)的活性顯著增強(p0.05)。TRALI兩次打擊細胞模型實驗顯示,儲存血小板制品中分離的PMPs可引發(fā)LPS活化的PMNs呼吸爆發(fā),導致HMVECs的損傷。 綜上所述,目前血庫血小板儲存條件(22℃,5天)下,血漿中PMPs數量和sCD40L含量升高。PMPs表面攜帶的sCD40L可能是在PMPs引發(fā)PMNs呼吸爆發(fā),進而誘導HMVEC損傷的效應分子之一。由此證明,輸注血庫儲存的血小板制品可能對TRALI的發(fā)生具有一定的影響。進一步研究PMP及所攜帶的sCD40L引發(fā)PMN呼吸爆發(fā)的機制,將為闡明TRALI的發(fā)病機制提供相關的實驗依據。
[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，
本文編號：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，
本文編號：2265166
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