本文關(guān)鍵詞：mir-142-5p在動(dòng)脈粥樣硬化斑塊中的表達(dá)及通過TGF-β2調(diào)控人巨噬細(xì)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(dòng)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(dòng)脈粥樣硬化是心血管疾病的一個(gè)主要的危險(xiǎn)因素,近年研究表明microRNAs在動(dòng)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(diào)控作用。動(dòng)脈粥樣硬化和心血管疾病的進(jìn)展中,microRNAs參與了包括內(nèi)皮功能的變化、血管平滑肌細(xì)胞增殖和遷移、巨噬細(xì)胞的功能以及泡沫細(xì)胞的形成。巨噬細(xì)胞參與先天免疫系統(tǒng),幫助清除凋亡細(xì)胞和去除細(xì)胞碎片產(chǎn)生的組織重構(gòu)和細(xì)胞壞死；巨噬細(xì)胞可以釋放炎性物質(zhì),影響動(dòng)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(xiàn)microRNAs不但參與了炎癥細(xì)胞的發(fā)育、分化,還參與調(diào)控炎癥細(xì)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(xì)胞胚胎干細(xì)胞方面都有研究,但在動(dòng)脈粥樣硬化方面的研究尚未見報(bào)道。研究目的(1)探討mir-142-5p在動(dòng)脈粥樣硬化斑塊中表達(dá)的變化；(2)進(jìn)一步探討mir-142-5p在人巨噬細(xì)胞中的表達(dá)；(3)探討mir-142-5p的靶基因并觀察其對(duì)人巨噬細(xì)胞凋亡功能的影響。方法1.1動(dòng)物模型的構(gòu)建實(shí)驗(yàn)選用8周齡的雄性ApoE-/-小鼠,在普食喂養(yǎng)3天后轉(zhuǎn)為高脂喂養(yǎng)；2周后給予右側(cè)頸動(dòng)脈套管術(shù)；給予套管高脂飲食8周,根據(jù)干預(yù)措施不同分為3組(12只/組)：空白對(duì)照組；穩(wěn)定斑塊組；易損斑塊組。易損斑塊組給予限制性應(yīng)激及噪音干預(yù)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(cè)將取材后的頸動(dòng)脈血管在冰上清理后送康城生物公司進(jìn)行microRNA芯片檢測(cè),篩查在易損斑塊表達(dá)變化較明顯的microRNA,明確mir-142-5p在動(dòng)脈粥樣硬化斑塊中的表達(dá)變化。1.3觀察mir-142-5p在血管壁不同細(xì)胞中的表達(dá),選擇變化明顯的細(xì)胞進(jìn)一步研究在人平滑肌細(xì)胞、內(nèi)皮細(xì)胞、巨噬細(xì)胞進(jìn)行培養(yǎng),用ox-LDL刺激,應(yīng)用RT-PCR技術(shù)比較mir-142-5p在不同細(xì)胞中的表達(dá)變化。1.4尋找mir-142-5p的靶基因通過數(shù)據(jù)庫(kù)及預(yù)測(cè)軟件預(yù)測(cè)mir-142-5p的靶基因：TGF-β2;通過脂質(zhì)體2000將mir-142-5p的inhibitor及mimics轉(zhuǎn)染進(jìn)入人巨噬細(xì)胞中,應(yīng)用Western-bolt及RT-PCR技術(shù)對(duì)靶基因TGF-β2進(jìn)行驗(yàn)證。1.5mir-142-5p及TGF-32干預(yù)對(duì)人巨噬細(xì)胞凋亡功能的影響應(yīng)用轉(zhuǎn)染技術(shù)將mir-142-5p的inhibitor、mimics及TGF-p2的inhibitor轉(zhuǎn)染進(jìn)入人巨噬細(xì)胞中,通過Annexin V-PE (Annexin V-PE Apoptosis Detection Kit)細(xì)胞凋亡檢測(cè)試劑盒在激光共聚焦顯微鏡下觀察其對(duì)人巨噬細(xì)胞功能的影響。1.5統(tǒng)計(jì)學(xué)分析兩組之間的差異比較采用獨(dú)立樣本t檢驗(yàn)；多組之間差異的比較采用了方差分析(ANOVA)。以上結(jié)果應(yīng)用了SPSS17.0軟件進(jìn)行統(tǒng)計(jì)分析。以P<0.05作為具有統(tǒng)計(jì)學(xué)意義。結(jié)果2.1mir-142-5p在斑塊中的表達(dá)MicroRNA芯片檢測(cè)結(jié)果顯示,mir-142-5p在穩(wěn)定斑塊組中的表達(dá)比空白對(duì)照組高6.8倍；mir-142-5p在易損斑塊組中的表達(dá)比空白對(duì)照組高2.7倍。2.2mir-142-5p在三種細(xì)胞中的表達(dá)培養(yǎng)人內(nèi)皮細(xì)胞、平滑肌細(xì)胞及巨噬細(xì)胞,并用ox-LDL50ng/ml刺激24h后提取(?)microRNA并應(yīng)用RT-PCR技術(shù)檢測(cè)mir-142-5p在三種細(xì)胞中的表達(dá),結(jié)果顯示內(nèi)皮及平滑肌細(xì)胞中mir-142-5p的表達(dá)在對(duì)照組與ox-LDL干預(yù)組間差異均低于1.5倍(P>0.05),而mir-142-5p在巨噬細(xì)胞ox-LDL干預(yù)組中的表達(dá)高于對(duì)照組5.4倍(P<0.05),與芯片結(jié)果趨勢(shì)符合。2.3mir-142-5p的靶基因預(yù)測(cè)應(yīng)用數(shù)據(jù)庫(kù)靶基因預(yù)測(cè)軟件預(yù)測(cè)mir-142-5p的靶基因可能為：TGF-β2;通過轉(zhuǎn)染技術(shù)將(?)nir-142-5p的inhibitor及rnimics轉(zhuǎn)染進(jìn)入人巨噬細(xì)胞中,提取蛋白及RNA, Western-bolt及RT-PCR的結(jié)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(yù)對(duì)人巨噬細(xì)胞凋亡功能的影響control組細(xì)胞基本無凋亡；與control組比較,ox-LDL組細(xì)胞凋亡增加(凋亡細(xì)胞率：6.83±0.17%,P<0.05)；mir-142-5p mimics+ox-LDL組(凋亡細(xì)胞率：5.27±0.19%)和TGF-β2inhibitor+mir-142-5p inhibitor+ox-LDL組(凋亡細(xì)胞率：1.38±0.27%)及TGF-β2inhibitor+ox-LDL組(凋亡細(xì)胞率：2.74±0.21%)細(xì)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(xì)胞最多(凋亡細(xì)胞率：12.31±0.22%,P<0.05)。凋亡細(xì)胞率=凋亡細(xì)胞數(shù)/總細(xì)胞數(shù)%。結(jié)論1mir142-5p在動(dòng)脈粥樣硬化斑塊中表達(dá)上調(diào)。2細(xì)胞學(xué)檢測(cè)顯示mir142-5p在人巨噬細(xì)胞中表達(dá)上調(diào),易損斑塊中表達(dá)增加明顯與芯片檢測(cè)的結(jié)果趨勢(shì)相符,具有抑制ox-LDL誘導(dǎo)的巨噬細(xì)胞凋亡作用。3TGF-β2可能是mirl42-5p的靶基因并參與調(diào)控人巨噬細(xì)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(dòng)脈粥樣硬化斑塊中的表達(dá)及通過TGF-β2調(diào)控人巨噬細(xì)胞凋亡功能的作用

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

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