摘要
目的探讨下肢静脉高压对血小板活化及静脉管壁重构的影响。方法将30只SD大鼠随机分成3组,各10只。A、C组应用髂静脉、股静脉缩窄法建立大鼠后肢静脉高压模型,分别以生理盐水、氯吡格雷25mg/(kg·d)饲喂;B组为假手术组。14d后,检测大鼠断尾出血时间;同时心脏取血,分离纯化血小板和单核细胞,应用流式细胞术检测血小板内部残留血小板因子4(PF4)以及单核细胞分化抗原115(CD115)-CD41双阳性比例,以评估血小板活化及血小板-单核细胞聚合比例;同时取大鼠股静脉组织进行Masson及CD115免疫组化染色。再取C57小鼠15只,随机分为3组,各5只,富血小板血浆(PRP)+凝血酶(thrombin)+氯吡格雷(clopi)组以氯吡格雷10mg/(kg·d)灌胃,PRP组及PRP+thrombin组以等量生理盐水灌胃,7d后内眦取血,离心获得PRP,将PBS及3组PRP分别与无血清DMEM制成体积分数0.05的条件培养液,分别与RAW264.7小鼠腹腔巨噬细胞Transwell共培养(PRP+thrombin组及PRP+thrombin+clopi组以激活剂活化),检测血小板对单核巨噬细胞迁移功能的影响。结果3组断尾出血时间比较,C组明显长于A组和B组,差异有统计学意义(F=243.10,P<0.01);A组与B组差异无显著性(P>0.05)。A组循环血小板内的PF4残余较B组和C组减少,血小板-单核细胞聚合百分比、股静脉管壁CD115表达及纤维化程度均明显升高,差异有显著性(F=26.40~243.10,P<0.01)。Transwell实验显示,PBS组细胞迁移数明显少于PRP组,PRP+thrombin组明显多于PRP+thrombin+clopi组,差异有显著性(F=25.70,P<0.01)。结论下肢静脉高压可以引起血小板异常活化及血小板-单核细胞聚合比例增高,并最终引起静脉管壁重塑。氯吡格雷可有效抑制静脉高压引起的血小板活化,同时可减轻管壁纤维化。
Objective To investigate the influence of lower limb venous hypertension on platelet activation and vein wall remodeling.Methods A total of 30 Sprague-Dawley rats were randomly divided into groups A,B,and C,with 10 rats in each group.The rats in groups A and C were used to establish a rat model of hindlimb venous hypertension by narrowing the iliac and femoral veins and were fed with normal saline and clopidogrel 25 mg/(kg·d),respectively,while those in group B were established as a sham-operation group.After 14 d,bleeding time was measured by tail cutting;cardiac blood was collected to isolate and purify platelets and monocytes,and flow cytometry was used to measure residual platelet factor 4(PF4)in platelets and CD115-CD41 double positive ratio of monocytes to evaluate platelet activation and the percentage of platelet-monocyte aggregates;femoral vein tissue was collected for Masson staining and CD115 immunohistochemical staining.A total of 15 C57 mice were randomly divided into platelet-rich plasma(PRP)+thrombin+clopidogrel(clopi)group,PRP group,and PRP+thrombin group,with 5 mice in each group.The mice in the PRP+thrombin+clopi group was given clopidogrel 10 mg/(kg·d)by gavage,and those in the PRP group and the PRP+thrombin group were given an equal volume of normal saline by gavage.After 7 d,blood was collected from the inner canthus and was then centrifuged to obtain PRP.PBS and PRP from the three groups were respectively added with serum-free DMEM to prepare a conditioned medium with a volume fraction of 0.05,and the 4 types of conditioned medium were respectively co-cultured with RAW264.7 mouse peritoneal macrophages in Transwell assay(the PRP+thrombin group and the PRP+thrombin+clopi group were activated by an activator)to evaluate the influence of platelets on the migration function of macrophages.Results Group C had a significantly longer bleeding time than groups A and B(F=243.10,P<0.01),while there was no significant difference between group A and group B(P>0.05).Compared with groups B and C,group A
作者
刘凯
颜京强
张鲲
齐浩山
赵俊成
李大林
LIU Kai;YAN Jingqiang;ZHANG Kun;QI Haoshan;ZHAO Juncheng;LI Dalin(Department of Vascular Surgery,Qingdao Municipal Hospital Affiliated to Qingdao University,Qingdao 266071,China)
出处
《青岛大学学报（医学版）》
CAS
2022年第4期615-620,共6页
Journal of Qingdao University(Medical Sciences)
基金
青岛市科技民生计划项目(13-1-3-4-n5h)。
关键词
静脉压
血小板活化
单核细胞
血管重塑
氯吡格雷
venous pressure
platelet activation
monocytes
vascular remodeling
clopidogrel
