脂多糖诱导血小板凋亡及 Toll 样受体4的介导作用

Role of Toll -like receptor 4 in lipopolysaccharide-induced platelet apoptosis

目的：探讨脂多糖（ lipopolysaccharide ， LPS）对血小板凋亡的诱导作用及血小板Toll样受体4（Toll－like receptor 4， TLR4）在LPS诱导的血小板凋亡中的作用。方法取健康志愿者的新鲜静脉血，分离富含血小板血浆，制备洗涤血小板，随机分为对照组（ C组）、LPS组（ L组）、TLR4单克隆抗体（ TLR4 monoclonal antibody ， TLR4mAb）预处理组（ P组）及凝血酶组（ T组）4组。各组均加入FcγRⅡ单克隆抗体封闭20 min；P组预先加入TLR4mAb，其余各组加入等量MTB缓冲液，室温孵育30 min；随后，L组及P组均加入LPS，T组加入凝血酶，C组加入等量的MTB，各组于室温下孵育30 min；裂解液裂解血小板，提取血小板蛋白，凋亡因子抗体芯片法检测各组凋亡相关蛋白的表达水平。采用芯片分析软件进行数据分析。结果人血小板可以表达多种凋亡相关蛋白。与C组比较，L组显示36种血小板凋亡相关蛋白的表达升高（ fold change ＞1畅50），包括促凋亡蛋白16种，抗凋亡蛋白8种，双向调控蛋白12种；与C组比较，T组可以诱导29种凋亡相关蛋白表达增高（fold change＞1．50），其中促凋亡蛋白15种，抗凋亡蛋白6种及双向调控蛋白8种；与L组比较，P组凋亡相关蛋白中有29种表达下调（fold change＜0．67），但与C组比较，仍有17种凋亡相关蛋白的表达增高（fold change＞1．50）。结论体外条件下，LPS可以通过内源性及外源性凋亡通路诱导人血小板发生凋亡，拮抗LPS－TLR4通路可能减少LPS诱导的血小板凋亡。

Objecitve To explore the effect of platelet apoptosis induced by lipopolysaccharide （LPS）and the role of Toll-like receptor 4 （TLR4） in this process.Methods Fresh venous blood was drawn from healthy human volunteers to isolate platelet -rich plasma （PRP ） and prepare washed platelets.The washed platelets were randomly divided into 4 groups：the control group （group C）, the LPS group （group L）, the TLR4mAb pretreatment group （group P） and the thrombin group （group T）. Each group was first incubated with the FcγRⅡ monoclonal antibody （ 20 min, room temperature ） . Platelets in group P were pretreated with TLR4mAb, while others with equal amount of MTB （30 min, room temperature ） .Then platelets in group L and P were incubated with LPS for 30 min at room temperature, while platelets in group T with thrombin and platelets in group C with MTB .Platelet lysate was prepared by cell lysis buffer .The expression of apoptosis proteins was detected by Human Apoptosis Antibody Array.Comparison between groups was carried out by using the RayBio ？ Analysis Tool. Results Human platelets can express many apoptosis -related proteins .Compared to group C , 36 kinds of apoptosis-related proteins were up -regulated in group L （fold change〉1.50）, including 16 pro-apoptotic members , 8 anti -apoptotic members and 12 proteins working both ways .29 kinds of proteins increased in group T （fold change 〉1.50） than group C, which included 15 pro-apoptotic members, 6 anti-apoptotic members and 8 proteins working both ways .29 kinds of apoptosis -related proteins in group P were lower than that in group L （fold change〈0.67）.But 17 kinds of apoptosis -related proteins in group P were still higher than those in group C （fold change〉1.50）.Conclusion Apoptosis of human platelet can be induced by LPS in vitro .This effect can be weakened by blocking the pathway of LPS-TLR4.