【摘要】：背景/目的：冠狀動脈粥樣硬化性心臟病(冠心病)是威脅我國人民生命健康的重要疾病之一,急性冠狀動脈綜合征(ACS)是冠心病的最嚴重并發(fā)癥,致殘率和致死率高。雖然早期診斷和再灌注治療對ACS患者的預(yù)后帶來很大改善,但是對于這種急性發(fā)病的疾病,臨床仍然缺乏有效預(yù)測ACS的生物標志物。血小板在ACS中發(fā)揮重要作用,高反應(yīng)性的血小板和ACS發(fā)生相關(guān),但是體外血小板功能實驗并不能完全反映體內(nèi)血小板功能,因此,準確反映體內(nèi)血小板激活狀態(tài)的標志物可能是預(yù)測ACS的重要方向。雖然已經(jīng)有文獻報道m(xù)icroRNAs (miRNAs)和血小板功能的研究,但是血漿外泌小體中miRNAs作為體內(nèi)血小板活性和預(yù)測急性心血管事件標志物的價值尚無研究。因此,本課題的研究目的是(1)通過對高脂飼料喂養(yǎng)的ApoE敲除(ApoE-/-)小鼠進行頸動脈雙狹窄手術(shù)(tandem stenosis, TS),建立動脈粥樣硬化血栓模型；(2)研究血栓形成之前血小板激活相關(guān)miR-223、miR-339和miR-21在血漿外泌小體中的變化；(3)以及這些miRNAs在血栓模型血漿外泌小體中和體外凝血酶激活的血小板源外泌小體中的表達水平一致性；(4)凝血酶激活的血小板源外泌小體在動脈粥樣硬化血栓形成中的作用,其對斑塊血管平滑肌細胞(SMCs)的調(diào)控機制。方法：ApoE-/-小鼠(n=55)高脂飼料喂養(yǎng)六周,進行雙狹窄(TS)手術(shù)(n=40)和sham手術(shù)(n=15),術(shù)后四周行血管超聲檢查TS組管腔血流情況,并經(jīng)左側(cè)頸靜脈取血,術(shù)后七周取材,行病理切片檢查血栓形成情況。分別將TS組發(fā)生血栓小鼠和sham組小鼠的術(shù)后四周血漿匯集,通過差速超速離心法提取血漿外泌小體,萃取其中的miRNAs,逆轉(zhuǎn)錄成cDNAs后進行熒光定量實時PCR,cel-miR-39作為內(nèi)參,以AACT法比較兩組間miR-223、miR-339和miR-21的表達差異。采集健康志愿者和野生型(WT)小鼠靜脈血,提取并純化血小板,分別用凝血酶(1U/ml)和PBS對照處理血小板,37℃孵育1小時,通過差速超速離心法提取血小板源外泌小體,比較凝血酶和PBS誘導的人和小鼠血小板源外泌小體中miR-233、miR-339和miR-21的表達差異。提取并分離VVT小鼠(4-6只)主動脈血管,進行主動脈SMCs培養(yǎng)。PKH26熒光素標記凝血酶誘導的血小板源外泌小體進行SMCs攝取外泌小體實驗,通過激光共聚焦顯微鏡分析SMCs攝取外泌小體的濃度、時間曲線。根據(jù)外泌小體在體外SMCs的代謝時間,檢測SMCs攝取外泌小體后的miR-223、miR-339和miR-21的變化。通過DAVID Tools用來分析這些miRNAs預(yù)測靶基因的富集通路,在RNA水平篩查這些變化的miRNAs可能調(diào)控SMCs的靶基因,并在蛋白水平通過激光共聚焦顯微鏡檢測免疫熒光表達量變化,并對體內(nèi)血栓節(jié)段進行驗證。通過Ki-67免疫熒光和TUNEL染色分析血小板源外泌小體對SMCs的增殖、凋亡的作用。結(jié)果：(1)TS組小鼠的血栓發(fā)生率37.5%(15/40),其中斑塊潰瘍占35%(14/40),斑塊破裂占2.5%(1/40)；血管壁內(nèi)膜中膜比在Sham組、TS組近端、TS組血栓段和TS組遠端分別為：0.119±0.029 vs 0.504±0.038 vs 1.078±0.072 vs1.195±0.116,p0.001;SMCs的Ki-67的熒光信號強度在sham組、TS近端、血栓節(jié)段和TS遠端的表達量分別為：1.002±0.027 vs 3.102±0.357 vs 2.837±0.063vs 3.120±0.403,p=0.001;TUNEL信號在sham組、TS近端、血栓節(jié)段和TS遠端的表達量分別為：0.997±0.095 vs 0.945±0.091 vs 5.472±1.077 vs 1.727±0.242,p=0.001；TS組小鼠頸動脈血管SMCs發(fā)生中膜向內(nèi)膜顯著遷移、細胞增殖顯著,而且血栓節(jié)段SMCs凋亡增加。(2)TS手術(shù)4周,頸動脈超聲明確沒有發(fā)生管腔閉塞,此時血漿外泌小體miR-223、miR-339和miR-21在TS組血栓形成和sham組的表達倍數(shù)分別為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)凝血酶對PBS處理人源血小板后,miR-233、miR-339和miR-21在血小板源外泌小體和碎片中的倍數(shù)變化分別為：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；凝血酶對PBS處理小鼠源血小板變化為：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攝取血小板源外泌小體隨濃度增加而攝取增多,攝取時間曲線符合拋物線模式：f(x)=-0.1345x2+3.2978x-1.0153,x(h),R2=0.9906,SMCs開始攝取外泌小體的時間為0.5 h,在12.25 h達到頂峰,在24 h外泌小體被細胞代謝完全。血小板源外泌小體處理SMCs24小時,細胞內(nèi)miR-223、miR-339和miR-21表達水平較PBS對照組升高倍數(shù)分別為：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-21參與的靶基因在MAPK通路中占42個,在總排名第二位,參與調(diào)控基因數(shù)占總通路總基因的2.9%,p=5.8x105,具有顯著相關(guān)性,其中PDGFRβ的mRNA表達水平在血小板源外泌小體處理的SMCs中顯著降低：0.67±0.19 vs 1.00±0.05,p=0.048；蛋白水平下降為：0.68±0.09vs 1.00±0.20,p=0.005。而且,sham組血管PDGFRβ熒光信號強度為基線,TS組節(jié)段SMCs的PDGFRβ表達顯著升高：3.03±0.42 vs 1.00±0.10,p0.001；但是,在血栓形成部位SMCs的PDGFRβ表達量顯著下降：2.15±0.10vs3.92±0.28,p=0.004。Ki-67陽性細胞比例在血小板源外泌小體預(yù)處理的SMCs中比例下降：4.15±1.91%vs 8.40±2.83%,p=0.047；TUNEL陽性細胞比例增加：13.35+1.45%vs 5.85±1.57%,p=0.025；提示血小板源外泌小體有效抑制PDGF-BB誘導的細胞增殖,且抑制PDGF-BB的抗細胞凋亡作用。結(jié)論：血漿外泌小體中miR-223、miR-339和miR-21水平升高與預(yù)測血栓形成相關(guān),其來源可能為血小板激活釋放增多。血小板源外泌小體抑制SMCs增殖、促進凋亡,可能通過轉(zhuǎn)運miR-223抑制PDGFRβ表達相關(guān)。模型血栓節(jié)段PDGFRβ水平表達下調(diào),進一步驗證血小板外泌小體在體內(nèi)動脈粥樣硬化血栓形成中的作用。本課題可能為血小板和SMCs之間信號傳導提供新途徑,血小板源外泌小體可能是預(yù)測動脈粥樣硬化血栓形成的潛在生物標志物。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R541.4

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2 譚明;血小板源外泌小體中microRNAs作為預(yù)測動脈粥樣硬化血栓形成的機制研究[D];北京協(xié)和醫(yī)學院;2016年

3 戴欣;microRNAs在胰腺癌相關(guān)性糖尿病中的表達及相關(guān)機制研究[D];上海交通大學;2013年

4 周可樹;MicroRNAs在慢性淋巴增殖性疾病中的表達及其在慢性淋巴細胞白血病預(yù)后中的意義研究[D];中國協(xié)和醫(yī)科大學;2010年

5 劉雯靜;胰腺癌特異性microRNAs的篩選及機制研究[D];北京協(xié)和醫(yī)學院;2011年

6 于雙妮;胰腺癌差異表達microRNAs的鑒定及功能研究[D];北京協(xié)和醫(yī)學院;2008年

7 熊薇;微泡microRNAs在肝癌診斷及發(fā)病機制中的作用研究[D];華中科技大學;2012年

8 陳娜;STAT3介導microRNAs調(diào)控的IL-9過表達在慢性淋巴細胞性白血病免疫調(diào)節(jié)機制中的研究[D];山東大學;2014年

9 高剛;氡致肺癌及肺損傷相關(guān)microRNAs的研究[D];中國疾病預(yù)防控制中心;2010年

10 張彩彩;硫化氫通過microRNAs調(diào)控血管新生的機制研究[D];復旦大學;2011年

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1 王倩;基于雙鏈特異性核酸酶介導的MicroRNAs檢測新方法的研究[D];華東理工大學;2016年

2 唐千捷;血漿microRNAs預(yù)測氯吡格雷抗血小板療效及心血管不良事件的研究[D];廣東藥科大學;2016年

3 楊翠云;基于信號放大的高靈敏腺苷與MicroRNAs電化學生物傳感器的制備及研究[D];西南大學;2016年

4 杜彥麗;microRNAs對黃牛和水牛朊病毒基因轉(zhuǎn)錄后調(diào)控研究[D];云南大學;2016年

5 楊丹鳳;以microRNAs為靶標結(jié)核病新型檢測方法的建立[D];吉林農(nóng)業(yè)大學;2016年

6 王曉鈺;基于microRNAs對上皮連接蛋白的調(diào)控探討升麻素抗過敏性炎癥復發(fā)的機制[D];南京中醫(yī)藥大學;2016年

7 張繼磊;炎癥相關(guān)因子和microRNAs在胃MALT淋巴瘤中發(fā)病機制的探討[D];第二軍醫(yī)大學;2012年

8 羅婷;microRNAs與慢性阻塞性肺疾病的研究進展[D];重慶醫(yī)科大學;2015年

9 羅曉燕;桃microRNAs鑒定及內(nèi)參基因的篩選[D];南京農(nóng)業(yè)大學;2013年

10 肖玲;胚胎干細胞RNAs與microRNAs相互作用網(wǎng)絡(luò)研究[D];華中師范大學;2012年

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