本文關鍵詞：mir-142-5p在動脈粥樣硬化斑塊中的表達及通過TGF-β2調控人巨噬細胞凋亡功能的作用，由筆耕文化傳播整理發(fā)布。

        研究背景冠狀動脈粥樣硬化性心臟病(冠心病,coronary heart disease, CHD)近年的發(fā)病率和死亡率逐年上升,成為威脅人類健康的重大主要原因。動脈粥樣硬化是心血管疾病的一個主要的危險因素,近年研究表明microRNAs在動脈粥樣硬化斑塊的形成及發(fā)展過程中參與并起著重要的基因調控作用。動脈粥樣硬化和心血管疾病的進展中,microRNAs參與了包括內皮功能的變化、血管平滑肌細胞增殖和遷移、巨噬細胞的功能以及泡沫細胞的形成。巨噬細胞參與先天免疫系統(tǒng),幫助清除凋亡細胞和去除細胞碎片產生的組織重構和細胞壞死；巨噬細胞可以釋放炎性物質,影響動脈粥樣硬化。在冠心病的發(fā)生及發(fā)展過程中,炎癥起著重要的作用。研究發(fā)現microRNAs不但參與了炎癥細胞的發(fā)育、分化,還參與調控炎癥細胞的活化以及各種炎癥因子的相互作用。mir-142-5p目前在腫瘤、免疫疾病、淋巴組織及紅細胞胚胎干細胞方面都有研究,但在動脈粥樣硬化方面的研究尚未見報道。研究目的(1)探討mir-142-5p在動脈粥樣硬化斑塊中表達的變化；(2)進一步探討mir-142-5p在人巨噬細胞中的表達；(3)探討mir-142-5p的靶基因并觀察其對人巨噬細胞凋亡功能的影響。方法1.1動物模型的構建實驗選用8周齡的雄性ApoE-/-小鼠,在普食喂養(yǎng)3天后轉為高脂喂養(yǎng)；2周后給予右側頸動脈套管術；給予套管高脂飲食8周,根據干預措施不同分為3組(12只/組)：空白對照組；穩(wěn)定斑塊組；易損斑塊組。易損斑塊組給予限制性應激及噪音干預4周(前兩周4h/天,后2周6h/天,5天/周)后,行麻醉處死小鼠采集血樣、收集樣本入液氮罐中。1.2microRNA芯片檢測將取材后的頸動脈血管在冰上清理后送康城生物公司進行microRNA芯片檢測,篩查在易損斑塊表達變化較明顯的microRNA,明確mir-142-5p在動脈粥樣硬化斑塊中的表達變化。1.3觀察mir-142-5p在血管壁不同細胞中的表達,選擇變化明顯的細胞進一步研究在人平滑肌細胞、內皮細胞、巨噬細胞進行培養(yǎng),用ox-LDL刺激,應用RT-PCR技術比較mir-142-5p在不同細胞中的表達變化。1.4尋找mir-142-5p的靶基因通過數據庫及預測軟件預測mir-142-5p的靶基因：TGF-β2;通過脂質體2000將mir-142-5p的inhibitor及mimics轉染進入人巨噬細胞中,應用Western-bolt及RT-PCR技術對靶基因TGF-β2進行驗證。1.5mir-142-5p及TGF-32干預對人巨噬細胞凋亡功能的影響應用轉染技術將mir-142-5p的inhibitor、mimics及TGF-p2的inhibitor轉染進入人巨噬細胞中,通過Annexin V-PE (Annexin V-PE Apoptosis Detection Kit)細胞凋亡檢測試劑盒在激光共聚焦顯微鏡下觀察其對人巨噬細胞功能的影響。1.5統(tǒng)計學分析兩組之間的差異比較采用獨立樣本t檢驗；多組之間差異的比較采用了方差分析(ANOVA)。以上結果應用了SPSS17.0軟件進行統(tǒng)計分析。以P<0.05作為具有統(tǒng)計學意義。結果2.1mir-142-5p在斑塊中的表達MicroRNA芯片檢測結果顯示,mir-142-5p在穩(wěn)定斑塊組中的表達比空白對照組高6.8倍；mir-142-5p在易損斑塊組中的表達比空白對照組高2.7倍。2.2mir-142-5p在三種細胞中的表達培養(yǎng)人內皮細胞、平滑肌細胞及巨噬細胞,并用ox-LDL50ng/ml刺激24h后提取(?)microRNA并應用RT-PCR技術檢測mir-142-5p在三種細胞中的表達,結果顯示內皮及平滑肌細胞中mir-142-5p的表達在對照組與ox-LDL干預組間差異均低于1.5倍(P>0.05),而mir-142-5p在巨噬細胞ox-LDL干預組中的表達高于對照組5.4倍(P<0.05),與芯片結果趨勢符合。2.3mir-142-5p的靶基因預測應用數據庫靶基因預測軟件預測mir-142-5p的靶基因可能為：TGF-β2;通過轉染技術將(?)nir-142-5p的inhibitor及rnimics轉染進入人巨噬細胞中,提取蛋白及RNA, Western-bolt及RT-PCR的結果均提示TGF-β2可能為mir-142-5p的靶基因。2.5Mir-142-5p及TGF-β2干預對人巨噬細胞凋亡功能的影響control組細胞基本無凋亡；與control組比較,ox-LDL組細胞凋亡增加(凋亡細胞率：6.83±0.17%,P<0.05)；mir-142-5p mimics+ox-LDL組(凋亡細胞率：5.27±0.19%)和TGF-β2inhibitor+mir-142-5p inhibitor+ox-LDL組(凋亡細胞率：1.38±0.27%)及TGF-β2inhibitor+ox-LDL組(凋亡細胞率：2.74±0.21%)細胞凋亡也較ox-LDL組少(P<0.05)；與其它組比較mir-142-5p inhibitor+ox-LDL組凋亡細胞最多(凋亡細胞率：12.31±0.22%,P<0.05)。凋亡細胞率=凋亡細胞數/總細胞數%。結論1mir142-5p在動脈粥樣硬化斑塊中表達上調。2細胞學檢測顯示mir142-5p在人巨噬細胞中表達上調,易損斑塊中表達增加明顯與芯片檢測的結果趨勢相符,具有抑制ox-LDL誘導的巨噬細胞凋亡作用。3TGF-β2可能是mirl42-5p的靶基因并參與調控人巨噬細胞凋亡。

    BackgroundMorbidity and mortality rate of coronary heart disease have been continous increasing in recent years and become serious threaten to human health. Atherosclerosis is a major risk factor of cardiovascular disease. Previous research have illustrated that microRNA play a key role as gene regulator in formation and development of atherosclerotic plaques. In development of atherosclerotic plaques and cardiovascular disease, microRNA participate in dysfunction of endothelium and macrophage, proliferation and migration of vascular smooth muscle cells and formation of foam cell.Research on expression status of mir-142-5p in atherosclerotic plaques has not been reported. Macrophage was involved in several progresses of innate immunity, including clearing apoptotic cell, avoiding cell apoptosis and tissue restructuring and release of inflammatory factors. Inflammatory play an important role in pathological process of CHD. MicroRNAs have been reported to participate in phylogenesis,differentiation and activiation of inflammatory cells. Previous research of mir-142-5p have focused on tumors, immunity diseases and stem cells. Research of mir-142-5p in CHD have not been reported at present.Research objectives(1) To discuss the changes of mir-142-5p expression level.(2)To investigate expression of mir-142-5p in macrophage further.(3)To explore target gene of mir-142-5p and the affect to apoptosis of macrophage.Methods1.1Construction of animal model8weeks old male ApoE-/-mice was used in present research. The mice was changed to high-fat diet after3days’basal diet for2weeks.Then we performed right common carotid artery tubulization. After high-fat diet for8weeks, the mice were divided to three groups(12mice each groups):control group, stabilized plague group, vulnerable plague group. The vulnerable plague group was given Pisaj syndrome noise interference for4weeks. Then all the mice were anesthesia and executed to obtain blood sample.1.2MicroRNA biochip detectionSend the flash-frozen carotid sample to Kangcheng-bio for a microRNA chip detection to identity the change of of mir-142-5p expression in atherosclerotic plaques.1.3The change of mir-142-5p expression in human macrophage.The expression level of mir-142-5p in ox-LDL stimulated cells was detected with RT-PCR technique.1.4Tracing for target gene of mir-142-5p.TGF-β2was predicted to be target gene of mir-142-5p. Inhibitor of mir-142-5p and mimics were transfected into human macrophage and detect the influence on TGF-β2by Western-bolt and RT-PCR.1.5mir-142-5p influences apoptosis of macrophage.Inhibitor of mir-142-5p mimics and inhibitor of TGF-β2were transfected into human macrophage. Function of human macrophage was observed by Annexin V-PE Apoptosis Detection Kit.1.6Statistics analysisData analysis was conducted with SPSS17.0. All data were presented as mean6standard error of the mean, and statistical comparisons were made with a paired t test and ANOVA tests. Differences were considered significant if P<0.05.Results2.1Expression of mir-142-5in plagueBiochip detection of microRNA results show expression level of mir-142-5p in stabilized plague group is6.8folds higher than blank control; expression level of mir-142-5p is2.7folds higher than vulnerable plagues group.2.2Expression of mir-142-5p in macrophage Human endothelial cells, smooth muscle cells and macrophage was cultured, then stimulated with ox-LDL50ng/ml for24hours before microRNA was extracted. RT-PCR was used to analyze expression of the3cell lines. In endothelial cells and smooth muscle cells, mir-142-5p expression of ox-LDL stimulated group decrease1.5folds compared with non-stimulated group. According to biochip detection result, mir-142-5p expression in macrophage cells is5.4folds higher than blank control.2.3Prediction of mir-142-5p target geneAs processed with database-based target gene prediction software, TGF-β2is the most probable target gene of mir-142-5p. Inhibitor of mir-142-5p and mimics was transfected into human macrophage cells. Protein and RNA were extracted and analyzed by western blot. Both results show TGF-β2is target gene of mir-142-5p.2.4mir-142-5p affects apoptosis of human macrophage cellsApoptosis of the blank control group was not observed. Cell apoptosis of ox-LDL group was increased(Apoptosis cell percentage:6.83±0.17%).Cell apoptosis of mir-142-5p mimics+ox-LDL group(Apoptosis cell percentage:5.27±0.19%);TGF-β2inhibitor mir-142-5p inhibitor+ox-LDL group (Apoptosis cell percentage:1.38±0.27%) and TGF-02inhibitor+ox-LDL group (Apoptosis cell percentage:2.74±0.21%) was also more slight than the other two groups. Cell apoptosis of N.C+ox-LDL group(Apoptosis cell percentage:6.49±0.25%) was also increased. Mir-142-5p restrains apoptosis of marque cells partly through TGF-β2.(P<0.05)Conclusions1. Expression of mir142-5p is at a high level in atherosclerotic plaques.2.mir142-5p is over expressed in human macrophage cells in CHD; Accords with the trend of the testing results of the chip, has the inhibitory effect of ox-LDL induced apoptosis.3. TGF-P2is target gene of mir142-5p and regultates apoptosis of human macrophage cells.

mir-142-5p在動脈粥樣硬化斑塊中的表達及通過TGF-β2調控人巨噬細胞凋亡功能的作用

中文摘要6-9英文摘要9-11符號說明12-13前言13-151 資料與方法15-262 結果26-283 討論28-354 結論35-365 附表附圖36-42參考文獻42-46綜述46-56    參考文獻53-56致謝56-57攻讀學位期間發(fā)表的學位論文目錄57-58附表58

  本文關鍵詞：mir-142-5p在動脈粥樣硬化斑塊中的表達及通過TGF-β2調控人巨噬細胞凋亡功能的作用，，由筆耕文化傳播整理發(fā)布。