本文關(guān)鍵詞： C1煤 宣威 肺腫瘤 實(shí)驗(yàn)裝置 動(dòng)物模型 發(fā)病機(jī)制　出處：《昆明醫(yī)科大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：[背景與目的]宣威地區(qū)(包括云南省宣威、富源、麒麟和沾益,貴州省盤(pán)縣,水城,六枝等),位于中國(guó)西南部,該地區(qū)擁有人口約310萬(wàn),是中國(guó)乃至世界肺癌的高發(fā)區(qū),有學(xué)者把當(dāng)?shù)胤伟┓Q之為宣威肺癌或珠江源肺癌。前期研究表明宣威地區(qū)肺癌死亡率的地理分布與晚二疊紀(jì)C1煙煤產(chǎn)出的地理分布在空間高度吻合,C1煙煤產(chǎn)出的區(qū)域肺癌發(fā)病率高。其中,宣威地區(qū)來(lái)賓鎮(zhèn)是當(dāng)?shù)胤伟┌l(fā)病率最高的鄉(xiāng)鎮(zhèn),高達(dá)128.31/100,000,是全國(guó)肺癌發(fā)病率的2-3倍；而同為宣威地區(qū)的文興鎮(zhèn),肺癌發(fā)病率卻只有5.83/100,000,遠(yuǎn)低于全國(guó)的發(fā)病水平。來(lái)賓鎮(zhèn)居民主要使用C1煙煤作為生活燃料,而文興鎮(zhèn)居民主要使用M30煤作為生活燃料。課題組前期對(duì)宣威來(lái)賓鎮(zhèn)C1煙煤的研究表明,C1煙煤中大量納米二氧化硅(Silicon dioxide,SiO2)賦存是導(dǎo)致當(dāng)?shù)胤伟└甙l(fā)的重要因素之一。而何興舟等對(duì)宣威地參區(qū)肺癌高發(fā)的研究也提示,宣威肺癌的高發(fā)與室內(nèi)空氣中的苯并芘(Benzoapyrene,BaP)污染相關(guān)。宣威肺癌高發(fā)的原因可能是由于物理和化學(xué)長(zhǎng)時(shí)間刺激和遺傳因素等共同造成。前期體外實(shí)驗(yàn)還表明,煤煙塵染毒Bease-2B細(xì)胞出現(xiàn)非編碼小RNA (microRNA,miRNA)的異常表達(dá)。miRNA被認(rèn)為在調(diào)控發(fā)育、腫瘤發(fā)生的過(guò)程中都起到重要作用。部分miRNA可能扮演著癌基因和抑癌基因的角色,部分miRNA通過(guò)調(diào)節(jié)原癌基因或以癌基因或抑癌基因形式參與腫瘤的形成。但燃煤燃燒產(chǎn)物的物質(zhì)成分以及對(duì)人體產(chǎn)生的影響等研究暫未見(jiàn)報(bào)道,目前研究局限于單一物質(zhì)的毒理學(xué)研究,尚缺乏能真實(shí)反映宣威當(dāng)?shù)厥覂?nèi)空氣污染的狀態(tài)裝置來(lái)建立相應(yīng)的動(dòng)物疾病模型；而動(dòng)物肺癌模型的建立能夠動(dòng)態(tài)研究發(fā)病過(guò)程,對(duì)研究宣威地區(qū)C1煙煤導(dǎo)致肺癌高發(fā)的機(jī)制具有重要的意義,可為進(jìn)一步尋找防治的靶點(diǎn)提供理論依據(jù)。本研究采用PM2.5濃度控制構(gòu)建基于宣威地區(qū)開(kāi)放式“火塘”的實(shí)驗(yàn)裝置；采用宣威地區(qū)不同燃煤(C1煙煤、M30煤)燃燒產(chǎn)物誘導(dǎo)F344大鼠構(gòu)建原發(fā)性肺癌模型,探討煙煤燃燒產(chǎn)物的成分對(duì)構(gòu)建動(dòng)物肺癌模型的影響；采用miRNAs芯片篩選異常表達(dá)的miRNAs,尋找肺癌發(fā)生過(guò)程中的靶基因。揭示宣威肺癌高發(fā)的分子機(jī)制,為當(dāng)?shù)胤伟┑姆乐翁峁⿲?shí)驗(yàn)依據(jù)。[方法]本研究分為三個(gè)部分：1、宣威地區(qū)燃煤特性調(diào)查和模擬室內(nèi)空氣污染實(shí)驗(yàn)裝置的建立：(1)、采用熱裂解儀對(duì)不同溫度下的C1煙煤與M30煤進(jìn)行熱裂解,使用GC-MS對(duì)熱裂解釋放的有機(jī)物進(jìn)行分析。(2)、通過(guò)模擬燃燒,采用大氣采樣儀對(duì)實(shí)驗(yàn)裝置內(nèi)的C1煙煤與M30煤燃燒產(chǎn)物的PM2.5采集于濾膜上,使用ICP-MS對(duì)濾膜上的無(wú)機(jī)成分進(jìn)行分析；(3)、采用風(fēng)扇以及單片機(jī)的聯(lián)合控制,模擬基于宣威地區(qū)開(kāi)放式“火塘”室內(nèi)空氣污染狀態(tài),構(gòu)建一個(gè)PM2.5相對(duì)恒定的封閉環(huán)境,用于實(shí)驗(yàn)動(dòng)物的空氣暴露染毒的實(shí)驗(yàn)裝置。2、構(gòu)建Cl煙煤燃燒產(chǎn)物染毒F344大鼠肺癌模型：使用自制的室內(nèi)空氣污染模擬實(shí)驗(yàn)染毒裝置對(duì)F344大鼠進(jìn)行C1煙煤和M30煤燃燒產(chǎn)物的染毒,建立F344大鼠肺癌模型；(1)將90只雌性F344大鼠隨機(jī)分為C1煙煤高劑量組、C1煙煤低劑量組、M30煤層高劑量組、M30煤層低劑量組、正常對(duì)照組,每組18只,進(jìn)行煤煙塵暴露；低劑量組每天分別暴露1小時(shí),高劑量組每天暴露2小時(shí)；(2)暴露總時(shí)間為180天,分別在30天、60天、90天、120天、150天、180天隨機(jī)處死F344大鼠,觀察將大鼠的肺組織肉眼外觀變化情況,將右肺以及氣管進(jìn)行甲醛固定后,包埋、切片、HE染色,觀察肺組織的變化情況以及成瘤情況,部分進(jìn)行戊二醛固定被電鏡用,左側(cè)肺葉進(jìn)行-80℃低溫速凍備基因芯片的檢測(cè)和western blot用；(3)使用透射電子顯微鏡觀察大鼠肺組織中超微結(jié)構(gòu)的變化。3、miRNA在C1煙煤燃燒產(chǎn)物染毒F344肺癌建模過(guò)程中的作用(1)采用第7代大鼠的miRCURY LNATM microRNA芯片對(duì)C1煙煤建模F344大鼠肺組織中MiRNA的表達(dá)情況進(jìn)行檢測(cè),篩選在建模過(guò)程中Mi-RNA的異常表達(dá)譜；(2)使用STEM法對(duì)異常表達(dá)的miRNA進(jìn)行趨勢(shì)分析,篩選持續(xù)異常表達(dá)的miRNA;(3)、使用mirbase, miranda和mirdb數(shù)據(jù)庫(kù)對(duì)持續(xù)異常表達(dá)的miRNA進(jìn)行靶基因信息的預(yù)測(cè),選取較為可信的靶基因；(4)采用qPCR法對(duì)異常的表達(dá)的miRNA進(jìn)行驗(yàn)證；(5)采用western blot對(duì)F344大鼠肺組織內(nèi)的靶蛋白進(jìn)行檢測(cè);(6)采用雙熒光素酶報(bào)告系統(tǒng)對(duì)靶基因進(jìn)行驗(yàn)證。[結(jié)果]1、(1)、煤熱解產(chǎn)物分析：①300℃時(shí)C1燃煤熱解產(chǎn)物有23種物質(zhì),其中PAHs類6種,占相對(duì)含量的6.08%；500℃時(shí)C1燃煤熱解產(chǎn)物有72種,其中PAHs類20種,占相對(duì)含量的33.70%；700℃時(shí)C1燃煤熱解產(chǎn)物有150種,其中PAHs類37種,占相對(duì)含量的37.81%；②300℃時(shí)M30燃煤熱解產(chǎn)物有12種物質(zhì),未見(jiàn)PAHs類產(chǎn)物釋放；500℃時(shí)M30燃煤熱解產(chǎn)物有63種,其中PAHs類17種,占相對(duì)含量的19.32%；700℃時(shí)M30燃煤熱解產(chǎn)物有126種,其中PAHs類38種,占相對(duì)含量的34.38%；③C1煙煤的熱解產(chǎn)物與M30熱解產(chǎn)物的有機(jī)成分不同,與M30煤相比,C1煙煤在低溫時(shí)就有PAHs的釋放,且相同溫度下C1煙煤釋放的PAHs的含量更高,結(jié)構(gòu)也更為復(fù)雜；(2)、PM2.5的成分分析：①C1煙煤的PM2.5中的鉻的含量(4.59±0.8ug/m3)比M30煤產(chǎn)鉻的含量(0.57±0.02ug/m3)高,兩者間差異有統(tǒng)計(jì)學(xué)意義(P0.05)；②C1燃煤的PM2.5中的鐵的含量(54.4±2.3ug/m3)比M30煤產(chǎn)鐵的含量(23.1±0.2ug/m3)高,兩者間差異有統(tǒng)計(jì)學(xué)意義(p0.05)；③C1燃煤的PM2.5中的硅的含量(3.74±0.21μg/m3)比M30煤產(chǎn)硅的含量(1.18±0.04μg/m3)高,兩者間差異有統(tǒng)計(jì)學(xué)意義(p0.05)(3)、通過(guò)PM2.5控制,成功構(gòu)建了室內(nèi)空氣污染的的實(shí)驗(yàn)裝置。2、(1)、Cl煙煤高劑量組成功誘導(dǎo)肺癌大鼠6只,誘癌成功率33.3%；M30煤層高劑量組誘導(dǎo)肺癌1只,誘癌成功率5.6%,余各組均未見(jiàn)肺癌形成,C1煙煤層高劑量組與各組間比較,差異具有統(tǒng)計(jì)學(xué)意義(p0.05);(2)、C1燃煤燃燒產(chǎn)物染毒F344大鼠的肺泡Ⅱ型細(xì)胞中的板層小體出現(xiàn)明顯減少,板層結(jié)構(gòu)消失,出現(xiàn)排空異常,呈空泡狀；超微結(jié)構(gòu)中主要以肺泡II型細(xì)胞的損傷為主。3、(1)、與正常對(duì)照組比,暴露30天有2條下調(diào)miRNA表達(dá)差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；暴露60天月有3條上調(diào)miRNA表達(dá)差異具有統(tǒng)計(jì)學(xué)意義(P0.05);暴露90天分別有3條上調(diào)miRNA和3條下調(diào)miRNA表達(dá)差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；暴露120天分別有9條上調(diào)miRNA和2條下調(diào)miRNA表達(dá)差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；暴露150天分別有10條上調(diào)miRNA和2條下調(diào)miRNA表達(dá)差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；暴露180天分別有17條上調(diào)miRNA和13條下調(diào)miRNA表達(dá)差異具有統(tǒng)計(jì)學(xué)意義(P0.05);(2)、STEM法分析發(fā)現(xiàn)miR-101B-3P、miR-433-5P、miR-872-3P、 miR-465-3P、miR-331-5P持續(xù)向上表達(dá),差異具有統(tǒng)計(jì)學(xué)意義(p0.05)；(3)、mirbase預(yù)測(cè)miR-872-3P有436個(gè)靶基因,miR-101B-3P有794個(gè)靶基因;miranda預(yù)測(cè)miR-872-3P有726個(gè)靶基因,miR-101B-3P有1083個(gè)靶基因,miR-465-3p有33個(gè)靶基因,miR-331-5p有82個(gè)靶基因；mirdb1預(yù)測(cè)miR-872-3P有51個(gè)靶基因,miR-101B-3P有296個(gè)靶基因；單個(gè)數(shù)據(jù)庫(kù)預(yù)測(cè)得到2359個(gè)靶基因；2個(gè)數(shù)據(jù)庫(kù)同時(shí)預(yù)測(cè)得到197個(gè)靶基因；3個(gè)數(shù)據(jù)庫(kù)同時(shí)預(yù)測(cè)得到18個(gè)靶基因；3個(gè)數(shù)據(jù)庫(kù)均預(yù)測(cè)有靶基因的miRNA只有miR-101B-3P和miR-872-3P;(4)、隨著造模時(shí)間的延長(zhǎng),C1煙煤燃燒產(chǎn)物高劑量組F344大鼠肺組織中miR-101B-3P和miR-872-3P持續(xù)表達(dá)升高；(5)、C1煙煤組染毒F344模型組的大鼠肺組織中均有Has2、Ing3、ogt、 Plcg1、Serpini1的表達(dá),Ing3和Serpini1蛋白的表達(dá)呈逐漸減低的趨勢(shì)；(6)、Ing3是miR-101b-3p的靶基因；Serpini1是miR-872-3p的靶基因；Has2不是miR-101b-3p的靶基因；Ogt不是miR-101b-3p的靶基因；Plcg1不是miR-101b-3p的靶基因。[結(jié)論]1、采用PM2.5濃度控制,可構(gòu)建宣威地區(qū)開(kāi)放式“火塘”致室內(nèi)空氣污染的實(shí)驗(yàn)裝置；2、C1煙煤燃燒產(chǎn)生的中鉻、鐵、硅和PAHs含量可能是造成宣威肺癌高發(fā)的重要因素之一；3、C1煙煤燃燒產(chǎn)物空氣暴露染毒可構(gòu)建原發(fā)性肺癌動(dòng)物模型；4、在C1煙煤燃燒產(chǎn)物構(gòu)建肺癌模型中,miR-101b-3p持續(xù)表達(dá),抑制了靶基因Ing3的功能,Ing3抑癌基因功能的抑制可能是C1煙煤誘發(fā)肺癌的重要機(jī)制；miR-872-3p持續(xù)表達(dá),抑制了靶基因Serpini1的功能,Serpinil基因功能可能是C1煙煤誘發(fā)肺癌的重要機(jī)制；Ing3和Serpinil可能是潛在的肺癌防治的靶點(diǎn)。
[Abstract]:Background and objective: Xuanwei area (including Yunnan province Xuanwei and Fuyuan, kylin and Zhanyi, Guizhou province Panxian City, six, etc.), located in the southwest of China, the region has a population of about 3 million 100 thousand, and the world is Chinese lung cancer incidence area, some scholars have called the local lung cancer Xuanwei lung cancer or the source of the Pearl River lung cancer. Previous study showed that the geographical distribution of lung cancer mortality in Xuanwei and Late Permian C1 bituminous coal output in space is highly consistent, regional lung cancer incidence rate is high. The output of C1 bituminous coal in Xuanwei, Laibin town is the local highest incidence of lung cancer in the township, up to 128.31/100000, is 2-3 times the national incidence of lung cancer; the same as the Xuanwei Wen Xing Zhen, the incidence of lung cancer is only 5.83/100000, far below the national level. The incidence of Laibin town residents mainly use C1 bituminous coal as the fuel of life, and the democracy to use M30 in Xing Zhen Coal as the fuel of life. Research on Xuanwei town of C1 bituminous ourprevious showed that a large number of nano silica C1 (Silicon dioxide, SiO2 in bituminous coal occurrence) is one of the important factors in the local high incidence of lung cancer. And He Xingzhou of Xuanwei. Areas of high incidence of lung cancer also suggest that high Xuanwei lung cancer and in indoor air the benzo (Benzoapyrene, BaP). The causes of pollution related Xuanwei high incidence of lung cancer may be due to physical and chemical stimulation for a long time and genetic factors caused. Previous in vitro experiments also showed that the coal dust exposure in Bease-2B cell non small RNA encoding (microRNA, miRNA) the abnormal expression of.MiRNA is considered in the regulation of development, the process of tumor the play an important role. MiRNA may play a part in oncogenes and tumor suppressor genes, miRNA by regulating oncogenes or by oncogenes or tumor suppressor gene Due to the formation of tumors. But in the form of coal combustion products and material composition of human influence research has not been reported at present, toxicology research study is confined to a single material, the lack of state apparatus can reflect the local Xuanwei of indoor air pollution to establish animal models of disease and establish corresponding animal model of lung cancer; can the dynamic of disease, is of great significance to study the mechanism of high incidence of lung cancer Xuanwei C1 bituminous coal which can provide a theoretical basis for further targets for prevention and treatment. This study uses the PM2.5 concentration control building experimental Xuanwei open "fire" based on the different coal in Xuanwei area; (C1 bituminous coal, M30 coal combustion products) induced primary lung cancer model of F344 rats, to explore the influence of coal combustion products components on the construction of animal lung cancer model; recovery Screening of abnormal expression of miRNAs chip miRNAs for lung cancer target gene. In the process of revealing the molecular mechanism of Xuanwei lung cancer, for prevention of local lung cancer. Methods provide experimental basis for this study is divided into three parts: 1, the establishment of air pollution experiment device to investigate characteristics of coal in Xuanwei and simulation room: (1), using thermal cracking apparatus on different temperature of C1 coal and M30 coal pyrolysis, organic GC-MS on the release of pyrolysis were analyzed. (2), through the simulation of combustion, the combustion products of atmospheric sampling collection of PM2.5 on the membrane of the experimental unit in the instrument C1 coal and M30 coal smoke and use ICP-MS to analyze the inorganic composition of membrane; (3), the joint control of fan and MCU, simulation of Xuanwei open fire "indoor air pollution status based on building a PM2.5 phase of constant closure The environment for the experimental animal experimental device of.2 air exposure exposure, construction of Cl bituminous coal combustion products in F344 rat model of lung cancer: using a self-made device for indoor air pollution exposure combustion products of C1 bituminous coal and M30 coal simulation experiment on F344 rats were established F344 rat model of lung cancer; (1) 90 female F344 rats were randomly divided into high dose group C1 C1 bituminous coal, coal seam M30 low dose group, high dose group, low dose M30 coal group, normal control group, 18 rats in each group were exposed to coal dust; low dose group were exposed to 1 hours a day, every 2 hours exposure to high dose group (2); the total exposure time for 180 days, respectively, in 30 days, 60 days, 90 days, 120 days, 150 days, 180 days were sacrificed F344 rats, observe the changes of rat lung tissue visual appearance, the right lung and trachea after formaldehyde fixation, embedding, sectioning, HE staining, observe the lung organization And the changes of tumor formation, were fixed in glutaraldehyde by electron microscope, the left lung of -80 low temperature frozen by gene chip detection and Western blot; (3) using transmission electron microscope to observe the changes of the ultrastructure of.3 in lung tissue of rats exposed to F344, miRNA in the product modeling process of the role of C1 in lung cancer bituminous coal combustion (1) using miRCURY LNATM microRNA chip seventh generation rats were carried out to detect the expression of MiRNA in lung tissue of C1 rats in the modeling of F344 bituminous coal, screening of abnormal expression of Mi-RNA in the process of modeling the spectrum; (2) using the STEM method to analyze the trend of the abnormal expression of miRNA and abnormal expression of miRNA continuous screening; (3), miRBase, Miranda and mirdb forecast database on abnormal expression of miRNA gene for the target gene, and selects the more credible; (4) the abnormal expression of miRNA in using qPCR method For verification; (5) was used to detect the target protein Western blot on lung tissue in F344 rats; (6) using the dual luciferase system of target genes were verified. The results of]1 (1), analysis of coal pyrolysis products: 300 degrees of coal pyrolysis product of C1 has 23 kinds of substances, including PAHs 6, accounting for the relative content of 6.08%; 500 degrees of coal pyrolysis products C1 72, PAHs 20, accounting for the relative content of 33.70%; 700 degrees of coal pyrolysis products C1 150, PAHs 37, accounting for the relative content of 37.81%; II 300 C M30 coal pyrolysis products 12 kinds of substances released no PAHs products; 500 C coal pyrolysis product of M30 in 63, PAHs Class 17, accounting for the relative content of 19.32%; 700 degrees of coal pyrolysis products M30 126, PAHs 38, accounting for the relative content of 34.38%; organic components of pyrolysis products and the heat M30 the hydrolysate of C1 bituminous coal 涓嶅悓,涓嶮30鐓ょ浉姣，

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