本文關(guān)鍵詞：miR-149調(diào)控矽塵誘導(dǎo)肺纖維化的初步研究，由筆耕文化傳播整理發(fā)布。

        煤工塵肺（Coal workers’pneumoconiosis，CWP）是在生產(chǎn)過程中長期吸入矽塵、煤矽塵或煤塵所導(dǎo)致的以肺組織不可逆性纖維化為主的疾病，主要包括矽肺、煤矽肺和煤肺，是我國最嚴重的職業(yè)病之一。由于發(fā)病機制并不完全清楚，目前尚無有效的治療方法。大量研究表明，矽塵導(dǎo)致的肺纖維化過程包括肺組織炎性損傷、肺組織結(jié)構(gòu)破壞以及伴有基質(zhì)細胞聚集的肺組織修復(fù)。其病理特征為肺組織成纖維細胞過度增殖，多種膠原蛋白在肺組織內(nèi)過多沉積。其發(fā)生發(fā)展涉及到大量炎性因子及致纖維化因子的表達以及相關(guān)基因的復(fù)雜網(wǎng)絡(luò)調(diào)控。近年來，microRNA（miRNA）作為一種非編碼小RNA因其廣泛的基因調(diào)節(jié)作用引起了科學(xué)界的關(guān)注。有研究表明miRNA在炎癥、組織發(fā)育與分化、細胞增殖、凋亡及組織修復(fù)過程中均發(fā)揮了重要作用，這些廣泛的生物學(xué)作用使其成為調(diào)節(jié)器官表型的關(guān)鍵分子及多種疾病的治療靶標(biāo)。目前有關(guān)miRNA與疾病之間關(guān)系的研究很多，尤其是腫瘤，如肺癌、肝癌、胃癌、前列腺癌、結(jié)直腸癌等，此外也包括心肌梗死、心衰、慢性腎病等。但是關(guān)于miRNA在塵肺病中的表達譜及功能學(xué)研究報道較少。因此，結(jié)合我國塵肺病的發(fā)病現(xiàn)狀，我們進行了煤工塵肺血清miRNA的測序分析，經(jīng)過生物信息學(xué)分析篩檢出煤工塵肺與對照表達差異的miRNA，并運用體內(nèi)試驗及體外實驗對煤工塵肺差異表達變化一致且最顯著的miRNA優(yōu)先進行了初步的功能學(xué)驗證，，探討這些miRNA在矽肺病發(fā)生發(fā)展中可能的分子機制，期望能將肺纖維化基因表達調(diào)控的網(wǎng)絡(luò)理解提高到一個新的水平，為找出特異性的血清miRNA作為煤工塵肺輔助診斷的生物標(biāo)志物奠定理論基礎(chǔ)，同時也為進一步研究預(yù)防和有效治療塵肺病提供線索。我們主要開展了兩個階段的研究：（1）煤工塵肺病人血清miRNA標(biāo)志物篩檢：即首先通過流行病學(xué)調(diào)查選擇病例和對照、SOLiD測序法篩選出差異表達的miRNA，利用miRWalk綜合數(shù)據(jù)庫對候選miRNA所調(diào)節(jié)的靶基因及信號通路進行了分析；（2）miR-149在SiO2粉塵所致肺纖維化中的功能學(xué)研究：在第一階段研究的基礎(chǔ)上，選擇其中一種差異表達明顯的miRNA（miR-149）進行了初步的功能學(xué)驗證。第一部分：煤工塵肺特異的血清miRNA篩檢目的：通過對CWP病人及接塵對照血清差異表達miRNA進行分析，發(fā)現(xiàn)影響CWP發(fā)生發(fā)展的關(guān)鍵miRNA，為提高塵肺病的早期診斷率及探索新的治療方法提供科學(xué)依據(jù)，也為進一步研究塵肺病發(fā)病機制提供線索。方法：（1）研究對象的選擇：2010年8月通過調(diào)查問卷收集某煤礦井下接塵男性工人的年齡、職業(yè)史（工齡、工種、防護措施等）、現(xiàn)病史、既往史、個人史等資料，并由專業(yè)職業(yè)病體檢機構(gòu)對接塵工人進行職業(yè)病體檢，拍攝高仟伏胸片及DR胸片，根據(jù)《塵肺病診斷標(biāo)準(zhǔn)》GBZ70-2009由職業(yè)病診斷醫(yī)師對研究對象進行塵肺病診斷。篩選出煤工塵肺病人27例（壹期、貳期、叁期病人各9例），對照9例，病例與對照在年齡、接塵年代、接塵工齡、工種、吸煙等方面相匹配；（2）全基因組miRNA測序：采集外周靜脈血并分離血清，同組血清等量混合，提取RNA，SOLiD測序儀檢測全基因組ncRNA表達，應(yīng)用SOLiD小RNA分析系統(tǒng)（錯配、過濾和比對）確定已知的miRNA、新miRNA及表達水平（拷貝數(shù)）；（3）靶基因預(yù)測及信號通路分析：將測序結(jié)果與miRNA數(shù)據(jù)庫比對，篩選出與對照組相比表達變化在2倍以上的miRNA，進一步確定在所有病例組變化方向一致的miRNA，通過miRWalk綜合數(shù)據(jù)庫對篩檢出的miRNA靶基因進行預(yù)測，并對其調(diào)控的信號通路進行分析。結(jié)果：全基因組測序法對接塵對照組、壹期、貳期、叁期CWP血清中ncRNA進行篩檢后，各組鑒定出與數(shù)據(jù)庫匹配的miRNA比例分別為39.9%、42.2%、36.4%、39.9%。以病例組與對照組比較差異大于2倍為篩選條件，壹期、貳期、叁期CWP篩選出的差異表達miRNA數(shù)量分別為32、55和39個，其中在所有病例組變化方向一致的miRNA共有5個，表達上調(diào)的有miR-25*、miR-9*；下調(diào)2倍以上的有miR-20b、miR-222、miR-149，其中以miR-25*及miR-149的變化較為顯著。文獻報道及靶基因分析顯示，這些miRNA可能調(diào)節(jié)著與細胞增殖、凋亡、炎癥、基因轉(zhuǎn)錄、細胞周期及信號轉(zhuǎn)導(dǎo)等相關(guān)基因的表達。結(jié)論：在病例組與接塵對照組血清中，共篩檢出5個變化方向一致的miRNA作為候選分子，其中表達明顯上調(diào)的為miR-25*、miR-9*，表達明顯下調(diào)的為miR-20b、miR-222、miR-149，這些miRNA可能調(diào)控了肺纖維化過程的關(guān)鍵信號通路。第二部分：miR-149在SiO2粉塵所致肺纖維化中的功能學(xué)研究目的：結(jié)合前期血清miRNA篩檢結(jié)果，通過體內(nèi)試驗及體外實驗研究miR-149在SiO2誘導(dǎo)的肺纖維化中的作用。方法：（1）體內(nèi)試驗：建立SiO2粉塵誘導(dǎo)的小鼠肺纖維化模型，通過qRT-PCR檢測miR-149的表達、免疫組織化學(xué)及Western blot等實驗技術(shù)觀察小鼠肺組織內(nèi)miR-149及炎癥因子IL-6的動態(tài)表達情況；（2）體外實驗：①ELISA法檢測研究對象血清中IL-6的表達情況；②應(yīng)用A549細胞及HBE細胞構(gòu)建SiO2粉塵處理的細胞模型，通過體外轉(zhuǎn)染miR-149mimics及inhibitor使其失調(diào)表達，應(yīng)用qRT-PCR檢測miR-149的表達、應(yīng)用Western blot及ELISA實驗觀察細胞表達IL-6的情況。結(jié)果：（1）與對照組相比，不同期別CWP病人血清中miR-149皆表達下調(diào)，IL-6的表達未發(fā)生明顯變化（P=0.684），但是隨著疾病進展，IL-6表達呈上調(diào)趨勢；（2）在SiO2粉塵誘導(dǎo)的小鼠肺纖維化模型中，免疫組織化學(xué)實驗結(jié)果顯示，25g/L SiO2粉塵處理小鼠后的第1周、第2周、第4周，其肺組織中的炎癥反應(yīng)較為明顯。在處理第1周，與對照組相比，生理鹽水組及粉塵組有多量的炎癥細胞浸潤；在第2周，生理鹽水組的炎癥反應(yīng)逐漸恢復(fù)，在第4周時炎癥已基本消失，但是粉塵組的炎癥反應(yīng)一直持續(xù)，且較重，到第4周時，肺組織內(nèi)肺泡壁增厚，肺泡結(jié)構(gòu)塌陷；Real-time PCR及Western blot實驗分別證實SiO2粉塵處理后的各時間點miR-149表達都明顯下調(diào)（P<0.000），而IL-6表達明顯上調(diào)；（3）在粉塵處理的細胞模型中，SiO2粉塵可明顯導(dǎo)致A549及HBE細胞凋亡增加，且A549細胞比HBE細胞更加敏感，用不同濃度SiO2粉塵處理細胞后其表達IL-6的水平明顯上調(diào)，存在明顯劑量效應(yīng)關(guān)系，而A549細胞除了具有劑量效應(yīng)關(guān)系外，隨著SiO2粉塵處理時間的延長，IL-6的表達水平也逐漸增加，還呈現(xiàn)出時間效應(yīng)關(guān)系；用50μg/ml SiO2粉塵處理A549細胞24h及150μg/mlSiO2粉塵處理HBE細胞12h后，HBE細胞內(nèi)miR-149的下調(diào)非常明顯，粉塵處理幾乎完全抑制了miR-149的表達（P<0.000），而在A549細胞內(nèi)miR-149的變化未發(fā)現(xiàn)統(tǒng)計學(xué)差異，但是可見到miR-149也呈下調(diào)趨勢（P=0.234）；A549細胞轉(zhuǎn)染miR-149相似劑后使IL-6的表達下調(diào)，而轉(zhuǎn)染抑制劑后上調(diào)了IL-6的表達水平。結(jié)論：通過體內(nèi)試驗及體外實驗我們發(fā)現(xiàn)：（1） SiO2粉塵處理可明顯導(dǎo)致小鼠肺組織的炎癥反應(yīng)；（2） miR-149的下調(diào)及IL-6的上調(diào)可能參與了SiO2粉塵處理后的炎癥、肺纖維化等相關(guān)過程；（3） miR-149可以負調(diào)控A549細胞表達IL-6的水平。

    Coal workers’ pneumoconiosis（CWP） is characterized by irreversible lungfibrotic diseases resulting from chronic dust inhalation（silica,silica and coal or coaldust）, which mainly includes the silicosis and coal pneumoconiosis. CWP is one ofthe most serious occupational disease in China with no effective therapies. Currentlythe pathogenesis of silicosis is still unknow. The numerous studies have shown thatthe processes of pulmonary fibrosis include inflammatory injury, tissue desdructionand abnormal tissue repair with matrix deposition. The pathological features showincreasing fibroblast proliferation and collagen synthesis in the lung tissue. And itinvolves in a large number of inflammatory cytokines and fibrosis factor expressionand regulation of related genes in complex networks. Recently, microRNAs（miRNAs） as small noncoding RNA molecules of1822nucleotides （nt） in length,play critical roles in various physiological processes such as inflammation, tissuedevelopment and differentiation, cellular apoptosis, proliferation, and tissue repair,which have emerged in the recent decade as key regulators of organ phenotypes andpotential targets for therapeutic interventions in multiple diseases. But there are fewstudies refer to miRNAs in the research of silicosis. Considering the prevalence forsilicosis in China, the present study was about the miRNA screening in serum ofCWP patients and healthy controls, and further functional studies both in vivo and in vitro were also conducted on the candidate miRNA. Hoping to find out thebiomarkers of diagnosis, progression and treatment of CWP, and further provide cluesto reveal the pathogenesis of CWP/silicosis and the roles for certain miRNAs in it.The dissertation is divided into two parts: analysis of serum genome-widemiRNAs for CWP in a case-control study and preliminary study of the role formiR-149in silica-induced pulmonary fibrosis.Part Ⅰ: Analysis of Serum Genome-wide miRNAs for CWP in a Case-ControlStudy.Objective: The key miRNAs which involved in pathogenesis and development ofCWP will be explored by screening the different expression of miRNAs in serumbetween cases and controls, so as to provide further clues to pathogenesis and newtherapies of pneumoconiosis.Methods:（1） The epidemiological information of the research subjects were obtainedby questionnaire, which included age, exposure history, jobs and other relatedepidemiological items. Health examinations including high KV chest X-ray and DRwere conducted by occupational professional agency. CWP patients were diagnosedaccording to the China national criteria for pneumoconiosis （GBZ70-2009）.One control group and three case groups which were CWP stage Ⅰ, stage Ⅱ and stageⅢ （n=9in each group） were mainly matched by age, exposure year, job, diseasehistory, smoking and so on.（2） Equal volume of sera from cases and controls in eachgroup were pooled for SOLiD sequencing-based miRNA profiling.（3） The keymiRNA target genes were primarily selected according to miRNA profiling resultsthat were blast to the known miRNA databases to identify the differently expressedmiRNAs with2-folds change, in which candidate miRNAs with the accordant changetrends in all of the case groups compared to the controls were identified. Genespotentially targeted by the selected miRNAs were identified by using the miRWalkdatabases available online.Results: Compared to the miRBase （15.0）,39.9%、42.2%、36.4%、39.9%knownprecursor miRNAs were identified in control, stage I, stage II and stage III of CWP, respectively. In these effective reads, the most abundant length was22nt size class.Using2-fold change as a cutoff level,32,55and39deregulated miRNAs wereidentified in each stage cases compared to controls, in which two up regulatedmiRNAs and three down regulated miRNAs with an accordant change in all of thecase groups compared to the paired controls were identified （miR-25*,miR-9*upregulated; miR-20b,miR-222and miR-149down regulated）. Target genes predictionand pathway analysis were then conducted for the candidate miRNAs, suggesting thatthe differentially expressed miRNAs regulate proliferation, apoptosis, inflammation,cell cycles, gene transcription and other biological process. Pathway analysis hasshown that candidate miRNAs regulated the key pathways in process of pulmonaryfibosis.Conclusion: Totally two up regulated and three down regulated miRNAs with anaccordant change direction were identified in all of case groups compared to thepaired control. And the candidate miRNA molecules may be involved in the keyprocesses for pulmonary fibrosis.Part Ⅱ: Preliminary Study on the Role of miR-149in Silica-induced PulmonaryFibrosis.Objective: Basing on the miRNA profile results, to explore the potential roles ofmiR-149in the pathogenesis of silica-induced pulmonary fibrosis.Methods: Studies were conducted both in vivo and in vitro to verify the preliminaryresults and to find new mechanisms of miR-149in regulating silica-inducedpulmonary fibrosis.（1） In vivo studies: The expression of miR-149was tested byReal-time PCR and inflammatory cytokine IL-6by immunohistochemistry andWestern blot assay in the lung tissues of silica-induced pulmonary fibrosis mice,.（2）In vitro studies:①The m odels ofSiO2exposed cells （A549and HBE） wereestablished. The A549cells were transfected with miR-149mimics and inhibitor, andthen the expression of IL-6in cells was tested by Western blot assay;②T heexpression of IL-6in CWP patients and paired controls was determined byEnzyme-linked immunosorbent assay. Results:（1） The serum expressions of miR-149in each stage of CWP patients weredown regulated compared to that of controls, but no significant differance was foundin expression of IL-6. However, it was found that the expression of IL-6was upregulated with the progress of CWP.（2） In the mice model, the pulmonary alveoli andinterstitial were infiltrated with amount of inflammatory cells after treated in week1in both silica-treated and saline-treated groups. The inflammation alleviatedgradually in saline-treated mice in2weeks, and was similar to that of controls. Butfor the silica-treated mice, the pulmonary alveolar wall were destoried and interstitialthickened obviously gradually. The expression of miR-149in lung tissues downregulated significantly in any check points（P<0.000）, while IL-6expression was upregulated obviously.（3） In the silica-treated cell models, the apoptosis of A549andHBE cells increased after silica-treated, especially for A549cells which was moresensitive to the silica challenge. For the A549cells, the expression of IL-6increasedin a silica-treated dose and time-dependant manner, while HBE cells only showsilica-treated dose-dependant effect. In addition, we found a significantly decrease formiR-149in HBE cells after12hours treated with the150μg/ml of silica（P<0.000）,the expression of miR-149was almost totally inhibited by the silica exposure, whileno significance was found inA549cells after24hours treated with the50μg/ml ofsilica（P=0.234）. But a decreased trend can be found in it. At last, we verified that thedysregulated expression of miR-149with miR-149mimics and inhibitor transfectioninfluenced the IL-6expression in A549cells. High expression of miR-149inhibitedIL-6expression. Reverse results were found after miR-149was inhibited.Conclusion:（1） Inflammatory reaction was induced after silica exposure in mice;（2）Down regulation of miR-149and up regulation of IL-6may involved in theinflammation and fibrosis process of silica-induced pulmonary fibrosis;（3） miR-149can inhibit IL-6expression in A549cells.

          miR-149調(diào)控矽塵誘導(dǎo)肺纖維化的初步研究

縮略語5-7中文摘要7-11Abstract11-14第一部分：煤工塵肺特異的血清 miRNA 篩檢15-35    前言15-17    調(diào)查內(nèi)容與方法17-20    結(jié)果20-26    討論26-30    參考文獻30-35第二部分：miR-149 在 SiO_2粉塵所致肺纖維化中的功能學(xué)研究35-64    前言35-37    材料與方法37-50    結(jié)果50-58    討論58-60    結(jié)論60-61    參考文獻61-64綜述64-79    參考文獻72-79碩士期間發(fā)表論文79-97致謝97

  本文關(guān)鍵詞：miR-149調(diào)控矽塵誘導(dǎo)肺纖維化的初步研究，由筆耕文化傳播整理發(fā)布。