血小板微粒及其攜帶的sCD40L在輸血相關(guān)急性肺損傷中作用的初步研究
發(fā)布時(shí)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(chǔ)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(xué)功能的促炎因子。在其刺激下,多形核中性粒細(xì)胞(polymorphonuclear neutrophil, PMNs)可快速移位至肺毛細(xì)血管部位,經(jīng)引發(fā)活化后產(chǎn)生呼吸爆發(fā),釋放大量的超氧陰離子和活性氧,導(dǎo)致組織損傷,誘發(fā)輸血相關(guān)急性肺損傷(TRALI)的發(fā)生。 本文主要研究血小板儲(chǔ)存過程中,儲(chǔ)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(dǎo)人肺微血管內(nèi)皮細(xì)胞(HMVECs)損傷的作用。重點(diǎn)討論儲(chǔ)存血小板制品中PMPs的性質(zhì)和生物學(xué)功能以及SCD40L的作用。 本研究取儲(chǔ)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(cè)血漿及PMPs中sCD40L的含量；抗體抑制實(shí)驗(yàn)評(píng)價(jià)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(dá)。Western Blotting檢測(cè)攜帶的CD40L蛋白分子的存在形式。以絕對(duì)細(xì)胞計(jì)數(shù)微球通過流式細(xì)胞術(shù)檢測(cè)血PMPs漿中數(shù)量。用活性氧特異性熒光探針二氫若丹明123(DHR)流式細(xì)胞術(shù)PMPs檢測(cè)呼吸爆發(fā)。建立(?)PMNs兩次打擊細(xì)胞模型,評(píng)價(jià)TRALI對(duì)PMPs介導(dǎo)的PMNs損傷的作用。HMVECs結(jié)果顯示,在血小板1-5天儲(chǔ)存過程中, 血漿中BC-PLTs含量逐漸遞sCD40L增,第3天達(dá)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(dá)到峰值。隨血小板儲(chǔ)存時(shí)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(qiáng),儲(chǔ)存第3天的血漿比第1天的血漿對(duì)PMNs的引發(fā)活性顯著提高(P0.05)。A-PLTs血漿對(duì)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(jié)構(gòu),表面攜帶CD40L且呈不均勻分 PMPs布。Western Blotting結(jié)果顯示PMPs所攜帶的CD40L分子量處于26-34kd之間。隨A-PLTs儲(chǔ)存時(shí)間的延長,血漿中PMPs數(shù)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(qiáng),儲(chǔ)存第3天比儲(chǔ)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(qiáng)(p0.05)。TRALI兩次打擊細(xì)胞模型實(shí)驗(yàn)顯示,儲(chǔ)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(dǎo)致HMVECs的損傷。 綜上所述,目前血庫血小板儲(chǔ)存條件(22℃,5天)下,血漿中PMPs數(shù)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(jìn)而誘導(dǎo)HMVEC損傷的效應(yīng)分子之一。由此證明,輸注血庫儲(chǔ)存的血小板制品可能對(duì)TRALI的發(fā)生具有一定的影響。進(jìn)一步研究PMP及所攜帶的sCD40L引發(fā)PMN呼吸爆發(fā)的機(jī)制,將為闡明TRALI的發(fā)病機(jī)制提供相關(guān)的實(shí)驗(yàn)依據(jù)。
[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(hào)：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(hào)：2265166
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