宣威地区C1烟煤燃烧产物诱导F344大鼠肺癌模型建立及其致病机制研究

发布时间：2018-01-24 18:09

  本文关键词： C1煤 宣威 肺肿瘤 实验装置 动物模型 发病机制　出处：《昆明医科大学》2016年博士论文　论文类型：学位论文

【摘要】：[背景与目的]宣威地区(包括云南省宣威、富源、麒麟和沾益,贵州省盘县,水城,六枝等),位于中国西南部,该地区拥有人口约310万,是中国乃至世界肺癌的高发区,有学者把当地肺癌称之为宣威肺癌或珠江源肺癌。前期研究表明宣威地区肺癌死亡率的地理分布与晚二叠纪C1烟煤产出的地理分布在空间高度吻合,C1烟煤产出的区域肺癌发病率高。其中,宣威地区来宾镇是当地肺癌发病率最高的乡镇,高达128.31/100,000,是全国肺癌发病率的2-3倍；而同为宣威地区的文兴镇,肺癌发病率却只有5.83/100,000,远低于全国的发病水平。来宾镇居民主要使用C1烟煤作为生活燃料,而文兴镇居民主要使用M30煤作为生活燃料。课题组前期对宣威来宾镇C1烟煤的研究表明,C1烟煤中大量纳米二氧化硅(Silicon dioxide,SiO2)赋存是导致当地肺癌高发的重要因素之一。而何兴舟等对宣威地参区肺癌高发的研究也提示,宣威肺癌的高发与室内空气中的苯并芘(Benzoapyrene,BaP)污染相关。宣威肺癌高发的原因可能是由于物理和化学长时间刺激和遗传因素等共同造成。前期体外实验还表明,煤烟尘染毒Bease-2B细胞出现非编码小RNA (microRNA,miRNA)的异常表达。miRNA被认为在调控发育、肿瘤发生的过程中都起到重要作用。部分miRNA可能扮演着癌基因和抑癌基因的角色,部分miRNA通过调节原癌基因或以癌基因或抑癌基因形式参与肿瘤的形成。但燃煤燃烧产物的物质成分以及对人体产生的影响等研究暂未见报道,目前研究局限于单一物质的毒理学研究,尚缺乏能真实反映宣威当地室内空气污染的状态装置来建立相应的动物疾病模型；而动物肺癌模型的建立能够动态研究发病过程,对研究宣威地区C1烟煤导致肺癌高发的机制具有重要的意义,可为进一步寻找防治的靶点提供理论依据。本研究采用PM2.5浓度控制构建基于宣威地区开放式“火塘”的实验装置；采用宣威地区不同燃煤(C1烟煤、M30煤)燃烧产物诱导F344大鼠构建原发性肺癌模型,探讨烟煤燃烧产物的成分对构建动物肺癌模型的影响；采用miRNAs芯片筛选异常表达的miRNAs,寻找肺癌发生过程中的靶基因。揭示宣威肺癌高发的分子机制,为当地肺癌的防治提供实验依据。[方法]本研究分为三个部分：1、宣威地区燃煤特性调查和模拟室内空气污染实验装置的建立：(1)、采用热裂解仪对不同温度下的C1烟煤与M30煤进行热裂解,使用GC-MS对热裂解释放的有机物进行分析。(2)、通过模拟燃烧,采用大气采样仪对实验装置内的C1烟煤与M30煤燃烧产物的PM2.5采集于滤膜上,使用ICP-MS对滤膜上的无机成分进行分析；(3)、采用风扇以及单片机的联合控制,模拟基于宣威地区开放式“火塘”室内空气污染状态,构建一个PM2.5相对恒定的封闭环境,用于实验动物的空气暴露染毒的实验装置。2、构建Cl烟煤燃烧产物染毒F344大鼠肺癌模型：使用自制的室内空气污染模拟实验染毒装置对F344大鼠进行C1烟煤和M30煤燃烧产物的染毒,建立F344大鼠肺癌模型；(1)将90只雌性F344大鼠随机分为C1烟煤高剂量组、C1烟煤低剂量组、M30煤层高剂量组、M30煤层低剂量组、正常对照组,每组18只,进行煤烟尘暴露；低剂量组每天分别暴露1小时,高剂量组每天暴露2小时；(2)暴露总时间为180天,分别在30天、60天、90天、120天、150天、180天随机处死F344大鼠,观察将大鼠的肺组织肉眼外观变化情况,将右肺以及气管进行甲醛固定后,包埋、切片、HE染色,观察肺组织的变化情况以及成瘤情况,部分进行戊二醛固定被电镜用,左侧肺叶进行-80℃低温速冻备基因芯片的检测和western blot用；(3)使用透射电子显微镜观察大鼠肺组织中超微结构的变化。3、miRNA在C1烟煤燃烧产物染毒F344肺癌建模过程中的作用(1)采用第7代大鼠的miRCURY LNATM microRNA芯片对C1烟煤建模F344大鼠肺组织中MiRNA的表达情况进行检测,筛选在建模过程中Mi-RNA的异常表达谱；(2)使用STEM法对异常表达的miRNA进行趋势分析,筛选持续异常表达的miRNA;(3)、使用mirbase, miranda和mirdb数据库对持续异常表达的miRNA进行靶基因信息的预测,选取较为可信的靶基因；(4)采用qPCR法对异常的表达的miRNA进行验证；(5)采用western blot对F344大鼠肺组织内的靶蛋白进行检测;(6)采用双荧光素酶报告系统对靶基因进行验证。[结果]1、(1)、煤热解产物分析：①300℃时C1燃煤热解产物有23种物质,其中PAHs类6种,占相对含量的6.08%；500℃时C1燃煤热解产物有72种,其中PAHs类20种,占相对含量的33.70%；700℃时C1燃煤热解产物有150种,其中PAHs类37种,占相对含量的37.81%；②300℃时M30燃煤热解产物有12种物质,未见PAHs类产物释放；500℃时M30燃煤热解产物有63种,其中PAHs类17种,占相对含量的19.32%；700℃时M30燃煤热解产物有126种,其中PAHs类38种,占相对含量的34.38%；③C1烟煤的热解产物与M30热解产物的有机成分不同,与M30煤相比,C1烟煤在低温时就有PAHs的释放,且相同温度下C1烟煤释放的PAHs的含量更高,结构也更为复杂；(2)、PM2.5的成分分析：①C1烟煤的PM2.5中的铬的含量(4.59±0.8ug/m3)比M30煤产铬的含量(0.57±0.02ug/m3)高,两者间差异有统计学意义(P0.05)；②C1燃煤的PM2.5中的铁的含量(54.4±2.3ug/m3)比M30煤产铁的含量(23.1±0.2ug/m3)高,两者间差异有统计学意义(p0.05)；③C1燃煤的PM2.5中的硅的含量(3.74±0.21μg/m3)比M30煤产硅的含量(1.18±0.04μg/m3)高,两者间差异有统计学意义(p0.05)(3)、通过PM2.5控制,成功构建了室内空气污染的的实验装置。2、(1)、Cl烟煤高剂量组成功诱导肺癌大鼠6只,诱癌成功率33.3%；M30煤层高剂量组诱导肺癌1只,诱癌成功率5.6%,余各组均未见肺癌形成,C1烟煤层高剂量组与各组间比较,差异具有统计学意义(p0.05);(2)、C1燃煤燃烧产物染毒F344大鼠的肺泡Ⅱ型细胞中的板层小体出现明显减少,板层结构消失,出现排空异常,呈空泡状；超微结构中主要以肺泡II型细胞的损伤为主。3、(1)、与正常对照组比,暴露30天有2条下调miRNA表达差异具有统计学意义(P0.05)；暴露60天月有3条上调miRNA表达差异具有统计学意义(P0.05);暴露90天分别有3条上调miRNA和3条下调miRNA表达差异具有统计学意义(P0.05)；暴露120天分别有9条上调miRNA和2条下调miRNA表达差异具有统计学意义(P0.05)；暴露150天分别有10条上调miRNA和2条下调miRNA表达差异具有统计学意义(P0.05)；暴露180天分别有17条上调miRNA和13条下调miRNA表达差异具有统计学意义(P0.05);(2)、STEM法分析发现miR-101B-3P、miR-433-5P、miR-872-3P、 miR-465-3P、miR-331-5P持续向上表达,差异具有统计学意义(p0.05)；(3)、mirbase预测miR-872-3P有436个靶基因,miR-101B-3P有794个靶基因;miranda预测miR-872-3P有726个靶基因,miR-101B-3P有1083个靶基因,miR-465-3p有33个靶基因,miR-331-5p有82个靶基因；mirdb1预测miR-872-3P有51个靶基因,miR-101B-3P有296个靶基因；单个数据库预测得到2359个靶基因；2个数据库同时预测得到197个靶基因；3个数据库同时预测得到18个靶基因；3个数据库均预测有靶基因的miRNA只有miR-101B-3P和miR-872-3P;(4)、随着造模时间的延长,C1烟煤燃烧产物高剂量组F344大鼠肺组织中miR-101B-3P和miR-872-3P持续表达升高；(5)、C1烟煤组染毒F344模型组的大鼠肺组织中均有Has2、Ing3、ogt、 Plcg1、Serpini1的表达,Ing3和Serpini1蛋白的表达呈逐渐减低的趋势；(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。[结论]1、采用PM2.5浓度控制,可构建宣威地区开放式“火塘”致室内空气污染的实验装置；2、C1烟煤燃烧产生的中铬、铁、硅和PAHs含量可能是造成宣威肺癌高发的重要因素之一；3、C1烟煤燃烧产物空气暴露染毒可构建原发性肺癌动物模型；4、在C1烟煤燃烧产物构建肺癌模型中,miR-101b-3p持续表达,抑制了靶基因Ing3的功能,Ing3抑癌基因功能的抑制可能是C1烟煤诱发肺癌的重要机制；miR-872-3p持续表达,抑制了靶基因Serpini1的功能,Serpinil基因功能可能是C1烟煤诱发肺癌的重要机制；Ing3和Serpinil可能是潜在的肺癌防治的靶点。
[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，

本文编号：1460669