血小板源外泌小体中microRNAs作为预测动脉粥样硬化血栓形成的机制研究

发布时间：2018-09-02 09:30

【摘要】：背景/目的：冠状动脉粥样硬化性心脏病(冠心病)是威胁我国人民生命健康的重要疾病之一,急性冠状动脉综合征(ACS)是冠心病的最严重并发症,致残率和致死率高。虽然早期诊断和再灌注治疗对ACS患者的预后带来很大改善,但是对于这种急性发病的疾病,临床仍然缺乏有效预测ACS的生物标志物。血小板在ACS中发挥重要作用,高反应性的血小板和ACS发生相关,但是体外血小板功能实验并不能完全反映体内血小板功能,因此,准确反映体内血小板激活状态的标志物可能是预测ACS的重要方向。虽然已经有文献报道microRNAs (miRNAs)和血小板功能的研究,但是血浆外泌小体中miRNAs作为体内血小板活性和预测急性心血管事件标志物的价值尚无研究。因此,本课题的研究目的是(1)通过对高脂饲料喂养的ApoE敲除(ApoE-/-)小鼠进行颈动脉双狭窄手术(tandem stenosis, TS),建立动脉粥样硬化血栓模型；(2)研究血栓形成之前血小板激活相关miR-223、miR-339和miR-21在血浆外泌小体中的变化；(3)以及这些miRNAs在血栓模型血浆外泌小体中和体外凝血酶激活的血小板源外泌小体中的表达水平一致性；(4)凝血酶激活的血小板源外泌小体在动脉粥样硬化血栓形成中的作用,其对斑块血管平滑肌细胞(SMCs)的调控机制。方法：ApoE-/-小鼠(n=55)高脂饲料喂养六周,进行双狭窄(TS)手术(n=40)和sham手术(n=15),术后四周行血管超声检查TS组管腔血流情况,并经左侧颈静脉取血,术后七周取材,行病理切片检查血栓形成情况。分别将TS组发生血栓小鼠和sham组小鼠的术后四周血浆汇集,通过差速超速离心法提取血浆外泌小体,萃取其中的miRNAs,逆转录成cDNAs后进行荧光定量实时PCR,cel-miR-39作为内参,以AACT法比较两组间miR-223、miR-339和miR-21的表达差异。采集健康志愿者和野生型(WT)小鼠静脉血,提取并纯化血小板,分别用凝血酶(1U/ml)和PBS对照处理血小板,37℃孵育1小时,通过差速超速离心法提取血小板源外泌小体,比较凝血酶和PBS诱导的人和小鼠血小板源外泌小体中miR-233、miR-339和miR-21的表达差异。提取并分离VVT小鼠(4-6只)主动脉血管,进行主动脉SMCs培养。PKH26荧光素标记凝血酶诱导的血小板源外泌小体进行SMCs摄取外泌小体实验,通过激光共聚焦显微镜分析SMCs摄取外泌小体的浓度、时间曲线。根据外泌小体在体外SMCs的代谢时间,检测SMCs摄取外泌小体后的miR-223、miR-339和miR-21的变化。通过DAVID Tools用来分析这些miRNAs预测靶基因的富集通路,在RNA水平筛查这些变化的miRNAs可能调控SMCs的靶基因,并在蛋白水平通过激光共聚焦显微镜检测免疫荧光表达量变化,并对体内血栓节段进行验证。通过Ki-67免疫荧光和TUNEL染色分析血小板源外泌小体对SMCs的增殖、凋亡的作用。结果：(1)TS组小鼠的血栓发生率37.5%(15/40),其中斑块溃疡占35%(14/40),斑块破裂占2.5%(1/40)；血管壁内膜中膜比在Sham组、TS组近端、TS组血栓段和TS组远端分别为：0.119±0.029 vs 0.504±0.038 vs 1.078±0.072 vs1.195±0.116,p0.001;SMCs的Ki-67的荧光信号强度在sham组、TS近端、血栓节段和TS远端的表达量分别为：1.002±0.027 vs 3.102±0.357 vs 2.837±0.063vs 3.120±0.403,p=0.001;TUNEL信号在sham组、TS近端、血栓节段和TS远端的表达量分别为：0.997±0.095 vs 0.945±0.091 vs 5.472±1.077 vs 1.727±0.242,p=0.001；TS组小鼠颈动脉血管SMCs发生中膜向内膜显著迁移、细胞增殖显著,而且血栓节段SMCs凋亡增加。(2)TS手术4周,颈动脉超声明确没有发生管腔闭塞,此时血浆外泌小体miR-223、miR-339和miR-21在TS组血栓形成和sham组的表达倍数分别为1.83±0.35vs 0.97±0.15,p=0.017；3.56±0.25 vs 1.03±0.15,p0.001；2.83±0.31 vs 0.98±0.18,p=0.01。(3)凝血酶对PBS处理人源血小板后,miR-233、miR-339和miR-21在血小板源外泌小体和碎片中的倍数变化分别为：2.05±0.48 vs 0.17±0.04,p=0.002；1.43±0.31 vs 0.28±0.07,p=0.003;1.40±0.17vs 0.25±0.02,p0.001；凝血酶对PBS处理小鼠源血小板变化为：1.34±0.10 vs0.45±0.02,p0.001;1.50±0.38 vs 0.40±0.05,p=0.007;1.41±0.23 vs 0.47±0.12,p=0.003。(4)SMCs摄取血小板源外泌小体随浓度增加而摄取增多,摄取时间曲线符合抛物线模式：f(x)=-0.1345x2+3.2978x-1.0153,x(h),R2=0.9906,SMCs开始摄取外泌小体的时间为0.5 h,在12.25 h达到顶峰,在24 h外泌小体被细胞代谢完全。血小板源外泌小体处理SMCs24小时,细胞内miR-223、miR-339和miR-21表达水平较PBS对照组升高倍数分别为：3.780±0.085 vs 1.027±0.041,p0.001；3.607±0.110vs 0.993±0.047,p0.001：5.620±0.269 vs 1.007±0.054,p0.001。KEGG富集通路分析提示miR-223、miR-339和miR-21参与的靶基因在MAPK通路中占42个,在总排名第二位,参与调控基因数占总通路总基因的2.9%,p=5.8x105,具有显著相关性,其中PDGFRβ的mRNA表达水平在血小板源外泌小体处理的SMCs中显著降低：0.67±0.19 vs 1.00±0.05,p=0.048；蛋白水平下降为：0.68±0.09vs 1.00±0.20,p=0.005。而且,sham组血管PDGFRβ荧光信号强度为基线,TS组节段SMCs的PDGFRβ表达显著升高：3.03±0.42 vs 1.00±0.10,p0.001；但是,在血栓形成部位SMCs的PDGFRβ表达量显著下降：2.15±0.10vs3.92±0.28,p=0.004。Ki-67阳性细胞比例在血小板源外泌小体预处理的SMCs中比例下降：4.15±1.91%vs 8.40±2.83%,p=0.047；TUNEL阳性细胞比例增加：13.35+1.45%vs 5.85±1.57%,p=0.025；提示血小板源外泌小体有效抑制PDGF-BB诱导的细胞增殖,且抑制PDGF-BB的抗细胞凋亡作用。结论：血浆外泌小体中miR-223、miR-339和miR-21水平升高与预测血栓形成相关,其来源可能为血小板激活释放增多。血小板源外泌小体抑制SMCs增殖、促进凋亡,可能通过转运miR-223抑制PDGFRβ表达相关。模型血栓节段PDGFRβ水平表达下调,进一步验证血小板外泌小体在体内动脉粥样硬化血栓形成中的作用。本课题可能为血小板和SMCs之间信号传导提供新途径,血小板源外泌小体可能是预测动脉粥样硬化血栓形成的潜在生物标志物。
[Abstract]:BACKGROUND/OBJECTIVE: Coronary atherosclerotic heart disease (CAHD) is one of the most important diseases threatening the life and health of Chinese people. Acute coronary syndrome (ACS) is the most serious complication of coronary heart disease with high disability and mortality. Platelets play an important role in ACS, and highly reactive platelets are associated with ACS. However, in vitro platelet function tests can not fully reflect the platelet function in vivo. Therefore, markers that accurately reflect the platelet activation state in vivo can be used. Although microRNAs (microRNAs) and platelet function have been reported in the literature, the value of microRNAs in plasma exosomes as a marker of platelet activity and predicting acute cardiovascular events has not been studied. In addition to (ApoE-/-) mice undergoing double carotid artery stenosis surgery (TS), atherosclerotic thrombosis models were established; (2) platelet activation-related microRNAs-223, microRNAs-339 and microRNAs-21 in plasma exosomes before thrombosis were studied; (3) and thrombin activation in plasma exosomes and in vitro of these microRNAs in thrombotic models. The expression level of platelet-derived exosomes was consistent; (4) The role of thrombin-activated platelet-derived exosomes in atherosclerotic thrombosis and its regulatory mechanism on plaque vascular smooth muscle cells (SMCs). Methods: ApoE-/-mice (n=55) were fed with high-fat diet for six weeks, and double stenosis (TS) surgery (n=40) and sham hands were performed. After operation (n=15), the blood flow in the lumen of TS group was examined by vascular ultrasound four weeks after operation, and the blood was taken from the left jugular vein. At the seventh week after operation, the thrombosis was examined by pathological section. RNA, reverse transcription of cDNAs, real-time fluorescence quantitative PCR, cel-microRNA-39 as internal reference, AACT method was used to compare the expression of microRNA223, microRNA339 and microRNA21 between the two groups. Differential ultracentrifugation was used to extract platelet-derived exosomes from human and mouse platelets. The expression of microRNAs-233, microRNAs-339 and microRNAs-21 in platelet-derived exosomes induced by thrombin and PBS was compared. The aortic vessels of VVT mice (4-6 mice) were isolated and cultured for SMCs. The concentration and time curve of SMCs uptake exosomes were analyzed by laser confocal microscopy. The changes of microRNAs-223, microRNAs-339 and microRNAs-21 after SMCs uptake were detected according to the metabolic time of exosomes in vitro. The changes of microRNAs may regulate the target genes of SMCs at RNA level, and the changes of immunofluorescence expression at protein level were detected by laser confocal microscopy, and the thrombus segments were verified in vivo. The effects of platelet exosomes on the proliferation and apoptosis of SMCs were analyzed by Ki-67 immunofluorescence and TUNEL staining. (1) The incidence of thrombosis in TS group was 37.5% (15/40), of which plaque ulcer was 35% (14/40) and plaque rupture was 2.5% (1/40); the ratio of intima-media to intima in SMCs group was 0.119 (+ 0.029) vs 0.504 (+ 0.038) vs 1.078 (+ 0.072) vs 1.195 (+ 0.116), and the fluorescence intensity of Ki-67 in SMCs group was 0.001. The expression of TUNEL signal in sham group, TS proximal end, thrombus segment and TS distal end were 1.002 + 0.027 vs 3.102 + 0.357 vs 2.837 + 0.063 vs 3.120 + 0.403, P = 0.001, respectively. The expression of TUNEL signal in sham group, TS proximal end, thrombus segment and TS distal end were 0.997 + 0.095 vs 0.945 + 0.091 vs 5.472 + 1.727 vs 1.727 + 0.242, P = 0.001, respectively. (2) Four weeks after TS operation, carotid artery ultrasonography showed that there was no lumen occlusion. At this time, plasma exosome microRNAs-223, microRNAs-339 and microRNAs-21 were expressed in thrombosis of TS group and in sham group. The expression multiples of microRNAs-223, microRNAs-339 and microRNAs-21 were 1.83 [0.35vs 0.97] 0.15, P = 0.017, respectively. 3.56 [+0.25 vs 1.03 [(1.03 [/0.05 [/0.48 vs 0.17 [/0.04, P = 0.002; 1.43 [/0.31 vs 0.31 vs 0.28 [/0.07, P = 0.07, P = 0.01. (3) After PBS treated human platele, the multiples of microRNA-233, microRNA-339 and microRNA-339 and microRNA-21 in Platele-derived exsecretory corpuscle and debris were 2.05 [/0.48 vs 0.48 vs 0.17 [/0.04, P = 0.04, P = 0.002; 1.43 [/0.31 vs 0.31 vs 0.28 [/0.28 [p0.001 The changes of thrombin on PBS-treated mice platelets were as follows: 1.34+0.10 vs 0.45+0.02, P 0.001; 1.50+0.38 vs 0.40+0.05, p=0.007; 1.41+0.23 vs 0.47+0.12, p=0.003. (4) SMCs uptake of platelet-derived exosomes increased with the increase of PBS concentration, and the uptake time curve conformed to the parabolic model: f(x) =-0.1345 x2+3.2978x-1.0153, x (h), R2 = 1.0153, R2 = 0.003. At 0.9906, SMCs began to take up exosomes at 0.5 h, reached the peak at 12.25 h, and were completely metabolized by cells at 24 h. The expression levels of intracellular microRNAs-223, microRNAs-339 and microRNAs-21 in SMCs treated with platelet-derived exosomes at 24 h were 3.780 (+ 0.085) vs 1.027 (+ 0.041), p0.001, 3.607 (+ 0.1100). The analysis of the enrichment pathway of 993+0.047, p0.001:5.620+0.269 vs 1.007+0.054, p0.001.KEGG indicated that 42 of the target genes involved in the MAPK pathway were microRNAs-223, microRNAs-339 and microRNAs-21, which ranked second in the total pathway. The number of genes involved in the regulation accounted for 2.9% of the total pathway genes, p=5.8x105. There was a significant correlation between the expression level of PDGFR beta mRNA in platelets. The expression of PDGFR beta in SMCs treated with exosomal bodies was significantly lower than that in SMCs treated with exosomal bodies: 0.67 (+ 0.19) vs 1.00 (+ 0.05), P = 0.048; the protein level decreased to: 0.68 (+ 0.09 vs 1.00 (+ 0.20), P = 0.005. Moreover, the fluorescence intensity of PDGFR beta in sham group was baseline, and the expression of PDGFR beta in TS group was significantly higher than that in thrombosis group: 3.03 (+ 0.42 vs 1.00 (+ 0.10), P 0.001. The expression of PDGFR-beta in SMCs decreased significantly: 2.15.10 vs 3.92.28, P = 0.004.Ki-67 positive cells decreased in SMCs pretreated with platelet-derived exosomes: 4.15 -1.91% vs 8.40 -2.83%, P = 0.047; TUNEL positive cells increased: 13.35 -1.45% vs 5.85 -1.57%, P = 0.025; suggesting that platelet-derived exosomes were effectively inhibited Conclusion: Increased levels of microRNAs-223, microRNAs-339 and microRNAs-21 in plasma exosomes are associated with predicting thrombosis, which may be due to increased platelet activation and release. The down-regulation of PDGFRbeta expression in thrombotic segments of the model further validates the role of platelet exosomes in atherosclerotic thrombosis in vivo. This study may provide a new pathway for signal transduction between platelets and SMCs, and platelet exosomes may be a potential predictor of atherosclerotic thrombosis. Biomarkers.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R541.4

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