【作者】 李光剑；

【导师】 黄云超；

【作者基本信息】 昆明医科大学 ， 外科学， 2013， 博士

【摘要】 [背景与目的]：中国云南省宣威地区是世界肺癌发病率最高的地区之一,女性肺癌发病率是中国其他地方的20倍。前期研究认为,这种高肺癌发病率与吸烟和工业污染等因素相关不明显,而与当地出产和使用的C1烟煤燃烧产物中含有的二氧化硅颗粒物有关。但是,之前的研究没有对C1烟煤燃烧产物中的二氧化硅进行定量,没有对燃烧产物中的二氧化硅颗粒物进行分离,这些二氧化硅颗粒物的粒径分布、表面特征和显微组份的依存关系是决定颗粒物对肺损伤的关键因素。另外,国际癌症研究机构己将结晶型二氧化硅规定为人类第一类致癌物质,是因吸入肺部后导致肺纤维化而诱发癌症。但是,这种自然产出的二氧化硅颗粒物吸入肺部后导致肺损伤甚至是肺癌的机制目前尚不清楚。我们模拟宣威地区女性人群暴露二氧化硅颗粒物的自然情况,进行长时间-低浓度经气管滴注工业合成的纳米二氧化硅颗粒物的大鼠诱癌试验,发现炎症反应是导致肺损伤的主要因素。但是,从炎症反应到癌症发生的分子生物学机制目前尚不清楚。最近的研究发现,一种非编码的小RNA (microRNA,miRNA)可能在炎症反应和癌症发生之间起“桥梁”作用。也就是炎症反应产生的炎性细胞因子能刺激miRNA表达水平升高,miRNA表达水平升高可通过扩大炎症效应而促进肿瘤发生。本研究的目的在于：(1)物理法从C1烟煤燃烧产物中分离二氧化硅颗粒物,研究这种自然产出的二氧化硅颗粒物在形貌特征,粒径分布和表面特征上与工业合成的二氧化硅和结晶型二氧化硅颗粒物有何差异；(2)模拟燃烧试验,间接法计算宣威地区农村女性人群从室内空气中吸入二氧化硅颗粒物的日暴露量均值,为体外细胞试验和动物试验提供实验依据；(3)通过基因芯片技术筛选与宣威地区非吸烟女性肺癌发生相关的miRNAs;(4)研究从C1烟煤燃烧产物中分离出的二氧化硅颗粒物对支气管上皮细胞炎性细胞因子分泌和miRNA表达的影响。初步探索自然产出的二氧化硅颗粒物可能的致癌机制。[方法]：(1)选取宣威肺癌高发区开采和使用的C1烟煤20份,以肺癌中等高发区开采和使用的烟煤(C2+1,K7)和肺癌低发区使用的烟煤(M30)为对照。从昆明医科大学第三附属医院病理科调取来自宣威地区(当地出生并居住生活3代以上)的非吸烟非小细胞女性肺癌患者术后病理组织标本20份。采用氟硅酸钾容量滴定法测量原煤和灰化后底灰中的二氧化硅含量,扫描电镜观察原煤和底灰中的二氧化硅颗粒物形貌,扫描电镜能谱分析二氧化硅颗粒物显微组份的依存关系,透射电镜观察单颗粒形貌。模拟当地用煤习惯和用煤量进行燃烧试验,空气污染监测仪收集煤烟尘,物理法从底灰和煤烟尘中分离二氧化硅颗粒物,激光粒度分析仪分析颗粒物的粒径分布,BET氮吸附比表面积仪测量颗粒物的比表面积。间接法计算宣威地区农村女性人群在使用C1烟煤时从室内空气中吸入二氧化硅颗粒物的日暴露量均值：(2)从昆明医科大学第三附属医院病理科调取来自宣威肺癌高发区的非吸烟非小细胞女性肺癌组织和对应非癌肺组织标本2对,基因芯片技术筛选癌组织中相对于非癌肺组织中差异表达的miRNAs和mRNAs, real time PCR技术对上调或下调最显著的miRNAs在20例来自宣威地区的非吸烟非小细胞女性肺癌组织中的表达量进行验证,生物信息遗传学预测上调最显著的miRNAs调控的靶基因。对癌组织中差异表达的1mRNAs进行基因功能富集分析；(3)体外细胞试验,以质量浓度和表面积浓度为衡量标准,台盼蓝抗法检测不同浓度刺激48h后支气管上皮细胞成活率变化,透射电镜观察100μg/mL二氧化硅颗粒物刺激支气管上皮细胞24h后,二氧化硅颗粒物在支气管上皮细胞内的亚细胞分布。根据细胞成活率试验和透射电镜观察结果,酶联免疫吸附法(ELISA)检测100μg/mL的二氧化硅颗粒物刺激支气管上皮细胞24h、48h后白介素8(IL-8)、γ干扰素(γ-IFN)、碱性成纤维细胞生长因子和成纤维细胞生长因子-2(bFGF/FGF-2)和血管内皮生长因子(VEGF)等细胞因子分泌变化,real time-PCR技术检测各时间点的miRNAs表达量变化。Pearson相关分析细胞因子分泌与miRNAs表达变化之间的关系。l结果1：(1)宣威肺癌高发区普遍使用的C1烟煤、煤燃烧产物中的二氧化硅含量显著高于肺癌低发区使用的M30烟煤和肺癌中等高发区使用的C2+1和K7烟煤(P<0.05)。各烟煤原煤中未发现游离的二氧化硅晶体,但可在C1烟煤燃烧产物(底灰和煤烟尘)中观察到二氧化硅晶体存在,同时在来自宣威肺癌高发区的女性肺癌癌组织中发现二氧化硅晶体存在。物理法可从C1烟煤燃烧产物中分离出二氧化硅颗粒物,这种自然产出的二氧化硅颗粒物形态不规整,表面不光滑,粒径分布多为纳米级,赋存有铝、钙、铁和锰元素。间接法测得宣威地区女性人群从室内空气中吸入二氧化硅颗粒物的日暴露量均值为2.25+0.35μg；(2)基因芯片分析结果显示,宣威地区非吸烟女性肺癌癌组织中有459个miRNAs表达水平相对于对应非癌肺组织中的表达水平显著不同,其中251个miRNAs显著上调(≥2倍),208个miRNAs显著下调(≥2倍)。其中miR-508-5P、miR1270和miR-501-5P上调最显著,miR-24-3P和miR-103a-3P下调最显著。通过real-time-PCR技术验证,miR-508-5P、miR1270和miR-501-5P在20例来自宣威地区的非吸烟女性肺癌癌组织中稳定高表达(P<0.05),而miR-24-3P和miR-103a-3P在癌组织中稳定低表达(P<0.05)。生物信息遗传学预测显著上调的miRNAs调控的靶基因多数与炎症应答有关。同时用基因芯片技术分析了27958个mRNAs,发现在宣威女性肺癌癌组织中有4263个基因显著上调,1922个基因显著下调,对差异表达基因进行基因功能聚类分析,参与免疫炎症应答的基因占79.4%；(3)和50-nm和结晶型二氧化硅颗粒物相比,C1烟煤燃烧产生的煤烟尘中分离出的二氧化硅颗粒物对支气管上皮细胞的成活率影响最大,透射电镜观察发现,这种自然产出的二氧化硅颗粒物更易穿透支气管上皮细胞膜而进入细胞内,所有的二氧化硅颗粒物均不能进入细胞核。自然产出的二氧化硅颗粒物和50-nm的二氧化硅颗粒物主要刺激支气管上皮细胞分泌IL-8和γ-IFN,而结晶型二氧化硅颗粒物主要刺激支气管上皮细胞分泌bFGF/FGF-2和VEGF。经100μg/mL自然产出的二氧化硅颗粒物刺激支气管上皮细胞24h后,miR-508-5P、miR1270和miR-501-5P的表达量与空白组相比分别上升了5、7和6倍。刺激48h后分别上升了7.5、10和10倍,显著高于50-nm二氧化硅组和结晶型二氧化硅组(P<0.0)。real-timePCR测定3个miRNAs共同调控靶基因PIK3AP的表达量,发现在自然产出的二氧化硅刺激组中显著低表达(P<001)。Pearson相关分析发现,刺激24h后,IL-8和y-IFN分泌量与3个miRNAs表达量之间的关联性为0.925和0.948, bFGF/FGF-2和VEGF分泌量与3个miRNAs表达量之间的关联性为-0.947和-0.964,刺激48h后,IL-8和y-IFN分泌量与3个1miRNAs表达量之间的关联性为0.978和0.933。bFGF/FGF-2和VEGF分泌量与3个]niRNAs表达量之间的关联性为-0.630和-0.732。[结论]：(1)宣威地区富含二氧化硅的C1烟煤与肺癌死亡率空间分布上高度吻合,燃烧产物和肺组织中均有二氧化硅颗粒物富集,颗粒物形态和显微组份上具有继承性。宣威地区C1烟煤中自然产出的纳米二氧化硅颗粒物存在“C1烟煤燃烧→烟煤中含有的纳米级二氧化硅颗粒物进入室内空气中→随空气悬浮颗粒物吸入肺内并沉积”的富集迁移路线；(2)宣威地区农村非吸烟女性肺癌可能是长时间-低浓度暴露燃煤中自然产出的二氧化硅颗粒物的结果；(3)宣威地区农村非吸烟女性肺癌人群可能存在：长时间-低浓度吸入燃煤中自然产出的二氧化硅颗粒物→肺部慢性非特异性炎症反应→炎性细胞因子大量释放→炎性细胞因子刺激多个miRNAs表达量上升→肺癌发生的致病途径。更多还原

【Abstract】 [Background and Objective]:China’s Xuanwei County in Yunnan Province have the worid’s highest incidence of lung cancer in non-smoking women-20times higher than the rest of China. Previous studies showed, this high lung cancer incidence may be associated with the silica particles embedded in the production combustion from C1coal, and no significant associated with smoking and industrial pollution. But, previous studies are not statistically quantified nor separated and characterization of silica particles in the production combustion from C1coal, these silica particles’size distribution, morphology, containing trace elements are important factors for the lung cancer epidemic. The International Agency for Research on Cancer (IARC) has classified crystalline silica was stated to be a class I carcinogen, because it can cause pulmonary fibrosis. But, we can’t know may be carcinogen mechanism of naturally occurring silica.We simulated the natural condition of women in Xuanwei County exposure to silica particles, for long time and low concentration transtracheal drip of silica nanoparticles in rat colon cancer, and found that the inflammation is the main form of lung injury. However, the molecular mechanism of from the inflammatory response to cancer is unclear, in recently studies, finding a no-code small RNA (microRNA, miRNA) may be serve as a bridge between inflammation and cancer. Also is the inflammatory cytokines produced from the inflammatory response can stimulate the miRNA expression up-regulated, and the miRNA expression up-regulated can expanding the inflammatory response and can promote the tumorigenesis. We study based on the following objects:(1) separateding the silica particles from the production combustion from C1bituminous, how unusual is the naturally occurring silica of morphology, size distribution, surface characteristics compared to the industrially occurring silica and crystalline silica by physical method.(2) Simulation of combustion test to calculation of daily total exposure of women living in the Xuanwei area to silica nanoparticles, which can provide a proof for cell experiment in vitro and animal experiment in vivo.(3) Gene chip technique screening miRNAs expression which associated with Xuanwei non-smoking female lung cancer.(4) Study the effect of naturally occurring silica caused miRNA expression and inflammatory cytokines excretion to bronchial epithelial cells, and preliminary exploration that the may be carcinogenic mechanism of naturally occurrlly occurring silica to human.[Methods]:(1)The20samples C1bituminous coal widely used in the high-incidence area of lung cancer in Xuanwei was selected as experiment group, while the C2+1, K7and M30bituminous coal that was mined and used in the low-incidence area of lung cancer in Xuanwei were selected as control group. Twenty paraffin-embedded cancer tissue samples from the non-smoking women with no small cell lung cancer who were born in Xuanwei and were at least the3rd generation of the family living there were collected from the department of pathology, the third affiliated hospital of Kunming medical university (tumor hospital of Yunnan province). Titrimetric potassium silicofluoride method was used to measure the content of silica in raw coal and its bottom ashes in20samples from the experimental group and control group. Scanning electron microscope (SEM) was used to observe the morphology of silica particles in C1bituminous coal and its bottom ashes, and scanning electron microscopy coupled with energy dispersive X-ray analyzer used to observe the morphology of silica particles in the bottom ashes and coal soot of C1bituminous coal as well as the lung cancer tissue from the non-smoking women in Xuanwei,and transmission electron microscope coupled with energy dispersive X-ray analyzer (TEM-EDX) was used to analyze the microscopic composition. The combustion experiments to simulate the consumption and cooking practice, soot was collected using air pollution monitoring equipment, separateding silica particles from soot by physical methods, particle size distribution was measured by a laser particle size analyzer, the surface areas of the particles were determined by BET nitrogen adsorption analysis, the women living in the Xuanwei area to silica nanoparticles every woman’s daily total exposure amount from indoor air was calculated by an indirect method.(2) Selecting2pairs cancer tissue and corresponding non-malignant lung tissue samples with no small cell and non-smoking female lung cancer from the department of pathology of the third affiliated hospital of Kunming medical university (tumor hospital of Yunnan province). Detecting miRNAs and mRNAs differentially expression between lung cancer tissues and their corresponding normal lung tissue, testing the miRNAs relative expression levels in20cases came from Xuanwei County by real time PCR technology which significant upregulate and downregulate in lung cancer tissue, biological information genetics predict the target genes controlled by significant upregulated miRNAs, gene ontology (GO) analysis that was used to categorize changes in mRNA expression in lung cancer tissues,(3) Respectively make the mass and surface area as a metric, assesssment the bronchial epithelial cells viability change exposed to silica after48-h at differential concentration, observing the subcellular distribution of silica nanoparticles in human brochial epithelial cells after24-h at100μg/mL by TEM, Enzyme Linked Immunosorbent Assay (ELISA) detecting Interleukin8(IL-8), y-interferon (y-IFN), Basic Fibroblast Growth Factor/Fibroblast Growth Factor2(bFGF/FGF-2) and Vascular Endothelial Growth Factor (VEGF) secretion changes of bronchial epithelial cells exposed to silica particles at100μg/mL after24-h and48-h, detecting the miRNAs expression levels by real-time PCR at0-h,24-h and48-h, Pearson correlation analysis the relationship between inflammatory cytokines secretion and the expression of miRNAs.[Results]:(1)The silica content in C1bituminous coal and its bottom ashes in the high-incidence area of lung cancer in Xuanwei was significently higher than that in C2+1, K7, and M30bituminous coal in the low-incidence area of lung cancer in Xuanwei(P<0.05). We can’t found the free silica particles in raw coal and can found free silica particles in combustion products (coal ashes and soot), and can observed the free silica particles in lung cancer tissue which come from high-incidence area of lung cancer in Xuanwei. We can separated the silica particles from combustion products (coal ashes and soot) by physical methods, these naturally occurring silica particles were mostly nanoscale, have various shape, large surface area, and contained such as iron,aluminium, calcium, and potassium. We calculated the daily silica nanoparticle exposure of women living in the Xuanwei area as2.25±0.35μg by indirectly method.(2) Genes chip analysis shows that have495miRNAs expression levels in non-smoking female lung cancer tissues differently from the corresponding non-malignant lung cancer tissues, which have251miRNAs significant up-regulared (≥2folds) and have208miRNAs significant down-regulated (<2folds), miR-508-5p, miR-1270, and miR501-5p significant up-regulated and miR-24-3p and miR-103a-3p significant down-regulated in non-smoking female lung cancer tissue of Xuanwei. Founding the miR-508-5p, miR-1270, and miR501-5p have a stablely up-regulate expression levels and the miR-24-3p and miR-103a-3p have a stablely down-regulate in20cases came from Xuanwei County by real time PCR technology, mostly target genes which controlled by significantly up-regulated miRNAs are contribute to inflammatory response through biological information genetics predict. Genes chip analysis27958mRNAs, finding4263genes significant up-regulated and1922genes significant down-regulated in non-smoking female lung cancer tissue of Xuanwei. Gene ontology (GO) analysis all differentially expression (DE) genes, finding have79.4%genes are contribute to inflammatory response.(3) The silica particles which separated from C1coal combustion production (soot) have a large affect to cell viability in human bronchial epithelial cells (BEAS-2B) compared to the50-nm silica and crystalline silica, TEM revealed that the naturally occurring silica particles more easily enter human bronchial epithelial cells, and all silica nanoparticles can’t enter cell nucleus. IL-8and y-IFN were released from BEAS-2B cells exposed to the naturally occurring silica particles and50-nm silica particles, bFGF/FGF-2and VEGF were released from BEAS-2B cells exposed to the crystalline silica. The miR-508-5p, miR1270and miR501-5p expression levels in human bronchial epithelial cells increased5,7and6times compared to blank control group exposed to naturally occurring silica particles at100μg/mL after24h, The miR-508-5p, miR1270and miR501-5p expression levels in human bronchial epithelial cells increased7.5,10and10times compared to blank control group exposed to naturally occurring silica particles at100μg/mL after48h, significant higher than the50-nm silica particles group and crystalline silica group (p<0.01). Detecting the target genes by three miRNAs control of Phosphoinositide3Kinase Adapter Protein (PIK3AP) expression levels by fluorescent probe, finding the expression level in naturallying occurring silica group significant lower than other group (p<0.01). Results of the correlation analyses indicate that the correlation between IL-8and开，啊-INF contains and three miRNAs expression level respectively are0.925and0.948, and the bFGF/FGF2and VEGF contain and three miRNAs expression level respectively are-0.947and-0.964after24-h, the correlation between IL-8and γ-INF contains and three miRNAs expression level respectively are0.978and0.933, and the bFGF/FGF2and VEGF contain and three miRNAs expression level respectively are-0.630and-0.732after48-h.[Conclutions]:(1) In Xuanwei, the incidence of lung cancer among non-smoking women is high in the area where silica-rich C1bituminous coal is produced. There are silica particles enriched in both the combustion products of Cl bituminous coal and the cancer tissue from the non-smoking women with non-small cell lung cancer, with similar morphology and microscopic composition. We hypothesize that the silica particles from combusted C1bituminous coal in Xuanwei are mixed with indoor air and inhaled along with other suspended particles.(2) The high lung cancer incidence in the Xuanwei area may thus result from low-dose, chronic exposure to naturally occurring silica nanoparticles over a longer time.(3) The high lung cancer incidence in the Xuanwei area may have pathogenic pathways: low-dose, chronic exposure to naturally occurring silica nanoparticles over a longer time, chronic nonspecific inflammation response can cause inflammatory cytokines released and stimulate the miRNAs expression up-regulate, and finally give rise to the lung cancer.更多还原