摘要
【目的】二磷酸腺苷受体P2Y_(12)介导的血小板活化是血小板活化及血栓形成的核心机制,本研究探讨囊性纤维化跨膜转导调节子(CFTR)对P2Y_(12)介导的血小板活化的影响。【方法】应用8~16周龄CFTR全基因敲除(Cftr^(-/-) )小鼠及野生型(Cftr^(+/+) )小鼠,分别制备胶原蛋白和肾上腺素诱导的肺栓塞小鼠模型。分离Cftr^(-/-) 和Cftr^(+/+) 小鼠外周血的血小板,采用免疫印迹技术检测P2Y_(12)、PI3K以及AKT总蛋白及磷酸化蛋白的表达。采用二磷酸腺苷(ADP)孵育人巨核细胞株Meg-01,检测ADP对CFTR和上述蛋白表达的影响。进一步应用特异性CFTR氯通道抑制剂CFTRinh-172与Meg-01细胞共孵育,检测其对CFTR和上述蛋白表达的影响。【结果】与Cftr^(+/+) 小鼠肺栓塞模型组相比,Cftr^(-/-) 鼠肺栓塞组织切片栓塞显著增加。Cftr^(-/-) 显著增加小鼠外周血血小板P2Y_(12)、磷酸化PI3K和AKT蛋白表达。P2Y_(12)全基因敲除小鼠血小板CFTR蛋白表达无明显改变,上述结果提示CFTR负性调控P2Y_(12)维持正常血小板活化功能。Meg-01细胞实验结果表明ADP浓度依赖性地诱导Meg-01细胞P2Y_(12)蛋白和PI3K以及AKT蛋白磷酸化增高,而CFTR蛋白表达水平逐渐降低。CFTR特异性阻断剂CFTRinh-172显著诱导Meg-01细胞P2Y_(12)蛋白表达增高。【结论】本研究揭示了血小板活化的一个新机制,即CFTR通过调控血小板P2Y_(12)蛋白表达影响血小板活化。
【Objective】Platelet activation mediated by adenosine diphosphate receptor P2Y_(12) is critical for platelet activation and thrombosis formation.The goal of the presented study was to investigate the role of CFTR in platelet activation mediated by P2Y_(12).【Methods】8-week-old CFTR knockout(Cftr^(-/-) )mice and age-matched wild-type(Cftr^(+/+) )mice were used to set up collagen and epinephrine-induced pulmonary embolism mouse models.The lung sections were prepared to observe the pulmonary embolism.The circulating platelets were isolated from peripheral blood of Cftr^(-/-) and Cftr^(+/+) mice,and were used to detect protein expressions of P2Y_(12),p-PI3K and p-AKT by western blot analysis.The above signaling proteins were also detected in human-derived megakaryocyte cell strains(Meg-01)treated with different concentration of adenosine diphosphate(ADP).CFTRinh-172,a selective CFTR chloride channel blocker was also applied to observe how CFTR affected the protein level of P2Y_(12) in Meg-01 cells【.Results】In the pulmonary embolism model,the embolization area in lung tissue sections from Cftr^(-/-) mice were significantly larger than that of Cftr^(+/+) mice.Compared with Cftr^(+/+) mice,the protein expression of P2Y_(12),p-PI3K and p-AKT in circulating platelets of Cftr^(-/-) mice was significantly elevated.However,no change was found in the protein expression of platelet CFTR in P2Y_(12) knockout mice,which suggested that CFTR might be in the upstream of P2Y_(12) in the regulating mechanism of platelet activation.In Meg-01 cells,ADP concentrationdependently induced P2Y_(12) protein expression but decreased CFTR expression.The incubation of Meg-01 cells with CFTRInh-172 could significantly increase P2Y_(12) expression.【Conclusions】This study revealed a new mechanism of platelet activation,that is,CFTR may regulate platelet activation by regulating P2Y_(12)-mediated signaling pathway.
作者
李俊
杨涵滟
韩慧
李梅
王冠蕾
LI Jun;YANG Han-yan;HAN Hui;LI Mei;WANG Guan-lei(Department of Pharmacology,Cardiac and Cerebral Vascular Research Center,Zhongshan School of Medicine,Sun Yat-sen University,Guangzhou 510080,China;VIP Healthcare Center,The Third Affiliated Hospital of Sun Yat-sen University,Guangzhou 510630,China)
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2021年第4期521-527,共7页
Journal of Sun Yat-Sen University:Medical Sciences
基金
国家自然科学基金(81773722,82073848)
广州市民生科技攻关计划(201803010092)。
