【作者】 张海英；

【导师】 金泰廙； 夏昭林；

【作者基本信息】 复旦大学 ， 卫生毒理学， 2007， 博士

【摘要】 矽肺是我国最为严重的职业卫生问题之一，其发病机制相当复杂至今尚未明确，可能涉及：矽尘诱导肺泡巨噬细胞产生各种细胞调节因子、诱发氧化应激损伤等，从而引起肺成纤维细胞增殖及细胞外基质的沉积。机体在应激状态下大量诱导合成应答蛋白，对细胞防御具有重要意义。目前关于应答蛋白与矽肺的相关研究较少，本课题采用离体实验和人群分子流行病学方法探讨应答蛋白与矽肺发生的关系。离体实验：大鼠肺泡巨噬细胞（AMs）和人胚肺成纤维细胞（HELF）组成体外模型。采用逆转录多聚酶链反应（RT-PCR）技术测定二氧化硅粉尘处理AMs的培养上清刺激HELF应答蛋白如热休克蛋白70（HSP70）各型（HSP70-1、HSP70-2和HSP70-hom）、热休克蛋白27（HSP27）、金属硫蛋白（MT）亚型1A、2A和血红素氧合酶-1（HO-1）以及与ECM合成、降解有关的基质金属蛋白酶-9（MMP9）及其组织抑制剂1（TIMP1）mRNA表达，观察其诱导表达水平的变化规律。同时，利用蛋白质组学的双向凝胶电泳分离技术与质谱鉴定技术（2-DE-MS）对模型中HELF蛋白表达进行分析并采用免疫印迹法（western blot）对其中的HSP27进行验证。一定范围内，HELF的HSP70-1、HSP70-2、HSP70-hom以及HO-1、HSP27等应答蛋白mRNA表达与二氧化硅（SiO₂）粉尘浓度呈现良好的剂量-效应关系和时间效应关系（P＜0.05），但不同的应答蛋白有不同的诱导动力学特征。各种应答蛋白被诱导表达达到峰值时间各有不同。MT1A、MT2A的mRNA表达虽能被染尘AMs上清诱导，但无明显的剂量-效应关系（P＞0.05）。MMP9、TIMP1能被明显诱导表达，但MMP9、TIMP1mRNA表达存在一定的时差，早期以MMP9增高为主，随着时间的延长，以TIMP1表达增加更为显著。2-DE-MS初步鉴定了经诱导的HELF的数个蛋白：HSP27、PeroxiredoxinⅡ亚型b、钙网蛋白-3和钙网蛋白-5类似物等。其中经western blot检测证实HSP27蛋白可被染尘AMs上清诱导表达，其表达水平随染尘剂量的增加而增加，与HSP27mRNA表达趋势基本一致。人群分子流行病学：采用病例-对照方法。选择中国西南某两大煤矿519例矿工为研究对象，其中接尘对照294例，确诊的矽肺患者225例（Ⅰ期134例、Ⅱ期73例、Ⅲ期18例）。采用工种接触数值估测（JEM）对累积粉尘暴露进行接触评估；对矿工的吸烟、饮酒等生活习惯、职业卫生习惯、职业危害认知状况、心理状况及营养状况等进行详细的问卷调查；进行肺功能检查并调查呼吸困难情况等对接尘矿工的健康效应进行评价；采用采用酶联免疫吸附测定（ELISA）方法对174例非吸烟人群（41名健康对照、133名矿工）血清中的MMP9／TIMP1进行测定；采用聚合酶链式反应-限制性内切酶片段长度多态性（PCR-RFLP）基因分型技术对519例矿工的MMP9（C-1562T）、HSP70-1（G190C）、HSP70-2（A1267G）和HSP70-hom（T2437C）基因进行基因分型。粉尘接触评估经单因素方差分析：JEM在病例组显著高于对照（P＜0.01）。病例组中Ⅰ期矽肺平均JEM：105.4±46.9，Ⅱ期矽肺平均JEM：114.3±40.3，Ⅲ期矽肺平均JEM：92.3±34.3，其别间差异有统计学差异（P＜0.01）。相关分析显示：JEM与矽肺分期呈明显的正相关（P＜0.01）。研究现场粉尘现况为：各项指标基本达标，但是个别工种平均浓度还有超标现象，总粉尘浓度甚至达到30％。提示还存在一定的隐患。对各期矽肺患者的肺通气功能测定并结合详细的个体调查进行分析：矽肺组肺功能参数中用力肺活量（FVC）、一秒钟用力呼气量（FEV1.0）、FEV1.0／FVC的实测值、实测值与预测值百分比均显著低于对照组（P＜0.05）；各项肺功能参数随着JEM分级的增加而呈下降的趋势（P＜0.05）；趋势卡方检验：肺功能异常类型发生率从高到低依次为限制性通气功能障碍、阻塞性通气功能障碍、混合性通气功能障碍。两分类Logistic回归分析，矿工肺功能障碍有统计学差异的影响因素按OR值从大到小依次排列为：Ⅱ+Ⅲ期矽肺（OR=12.557）、Ⅰ期矽肺（4.863）、BI＜400（2.971）、BI600～（2.504）、JEM150～（2.390）、BI400～（2.300）、慢支（1.715）。174例非吸烟人群（健康对照41例，接尘工人64例，矽肺患者69例）血清MMP9及TIMP1检测结果经秩和检验：与健康对照组比较，接尘工人血清中MMP9增加较为明显（P＜0.05），而TIMP1增加的差异没有统计学意义（P＞0.05），各期矽肺的MMP9、TIMP1浓度呈增加趋势，而MMP9／TIMP1比值呈下降趋势，且与健康对照组相比，差异有统计学意义（P＜0.05）。经JEM分级与年龄分级校正，与接尘对照组比较，Ⅰ期矽肺患者的TIMP1升高（P＜0.05），Ⅱ期矽肺患者MMP9／TIMP1比值增加（P＜0.05）以及Ⅲ期矽肺患者的MMP9、TIMP1升高（P＜0.05），而MMP9／TIMP1呈下降趋势（P＜0.01）。基因分型结果经卡方检验：HSP70-2（A1267G）等位基因频率在矽肺各期间分布差异有边缘统计学意义（P=0.046），G位点的携带率在Ⅱ+Ⅲ期矽肺中明显高于Ⅰ期矽肺和接尘对照组（P＜0.05）。Ⅱ+Ⅲ期矽肺的HSP70-hom（T2437C）的突变纯合子（C／C）携带率高于接尘对照组和Ⅰ期矽肺（8.8％vs2.4％和3％），在相同JEM分级和相同的布莱克曼吸烟指数（BI）分级，携带突变纯合基因型的个体发生矽肺的风险明显高于杂合子携带个体与野生纯合子携带个体（P＜0.05）。MMP9-1562C／C野生纯合子基因型与HSP70-2G位点携带者在Ⅱ+Ⅲ期矽肺中的分布与对照和Ⅰ期矽肺组比较差异有临界差异（P=0.087）；JEM（150～）水平下，HSP70-hom2437C突变位点的携带者中，其分布在矽肺与对照组比较也表现出临界差异（P=0.094）。吸烟和基因间的交互作用分析没有发现统计学差异（P＞0.05）。LDA分析显示HSP70-1（190）、HSP70-2（1267）和HSP70-hom（2437）三对等位基因处于高度连锁不平衡；PHASE软件进行单倍型分析：HSP70-1，HSP70-2和HSP70-hom的190，1267和2437等位基因构成的G-A-T和G-G-T单倍型在Ⅱ+Ⅲ期矽肺中明显增加，与Ⅰ期矽肺和接尘对照相比差异均有统计学意义（P＜0.05）。综上所述，HELF应答蛋白HSP70各型（HSP70-1、HSP70-2和HSP70-hom）、HSP27、HO-1以及MMP9／TIMP1mRNA的表达能被染SiO₂粉尘的AMs上清诱导并呈现一定的剂量-反应和时间-效应关系，但各自存在不同的诱导动力学特征，提示可能是因为被研究基因表达的调控序列存在差异；不同应答蛋白表达存在时相差，交错对细胞起到保护作用。MMP9／TIMP1mRNA表达时相差异提示早期以MMP9表达及活性增加，基质降解为主；TIMP1出现对MMP9起拮抗作用，随着MMP9／TIMP平衡打破，胶原沉积。HSP27、PeroxiredoxinⅡ及钙网蛋白等与SiO₂引起的氧化应激密切相关。预防矽肺的发生除了通过控制作业场所的粉尘浓度，同时做好个体防护，减少粉尘暴露；加强矿工职业卫生教育和健康促进，识别易感人群（如HSP70-hom2437C的C／C基因型、以及HSP70-1，HSP70-2和HSP70-hom的G-G-T、G-A-T单倍型携带者）。为达到WHO消除矽肺的目标而奋斗。更多还原

【Abstract】 Silicosis has long been one of the most serious and prevalent occupational healthproblems in China for its high morbidity and mortality. Although their pathogenesis remainsincompletely understood, some evidence suggest the participation of cytokines produced byAMs and oxidative stress induced by silica leading to the fibroblast proliferation andextracellular matrix（ECM） deposition. The heat stress proteins （HSPs） can be induced andplay important role for the cell protection as a result of various mechanical or chemicalstresses. A little is known about relation between HSPs and silicosis,the aim of this study is toexplore the roles of response proteins on occurrence of silicosis by in vitro experiments_andmolecular epidemiology.Sprague-Dawley （SD） rat alveolar macrophages （AMs） and human embryonic lungfibroblasts （HELF） was formed into an in vitro model. The mRNA levels of heat stressproteins（HSPs）, such as HSP70-1, HSP70-2,HSP70-hom, HSP27, metallothionein （MT）1A、MT2A, haemoxygenase-1（HO-1） and the reported factors related to the synthesis ordegradation of ECM, metalloproteinases-9（MMP9） and tissue inhibitor ofmetalloproteinases-1（TIMP1） of HELF stimulated by supernatants of AMs exposed to silicawere measured by RT-PCR technology. At the same time, identification of proteins of treatedHELF was conducted by two-dimensional polyacrylamide gel electrophoresis（2-DE） andmatrix assisted laser desorption-ionization-time of flight massspectrometry（MALDI-TOF-MS）. One of identified proteins,HSP27,was further validated byWestern blot assay.It was shown that there is good dose-effect and time-effect relationships betweenHSP70-1, HSP70-2, HSP70-hom, HSP27, HO-1, MMP9 and TIMP1 mRNA levels andconcentration of silica and supernatants exposure time （P＜0.05）. Different response proteinshad their own induction dynamic characters, and the time they reach the peak expression wasdiversity. It is not found dose-effect relationship between the MT1A, MT2A mRNA levels andthe concentration of silica, while they can be induced by the supernatants（P＞0.05）. MMP9,TIMP1 mRNA can be obviously induced and MMP9 mRNA levels increased earlier thanthose of TIMP1, but TIMP1 increased more obviously than MMP9 with time continuing.Several proteins in induced HELF were identified by 2-DE-MS: PeroxiredoxinⅡ isoform b, heat shock protein 27, a protein similar to calreticulin isoform 3, a protein similar tocalreticulin isoform 5. HSP27 was further validated by western blot and the results showedthat it increased with the concentration of silica increasing. The change tendency ofHSP27mRNA and protein expression was consistent.Epidemiologic investigation: Case-control study was applied in our study. Weinvestigated 519 workers from two large coal plants in Southwestern China, including 225patients with silicosis （Ⅰ: 134;Ⅱ, 73;Ⅲ, 18） and 294 control miners（self-reported to beHan descent, the primary ethnic group in China） with similar coal mine dust-exposurehistories but without apparent pulmonary disease or inflammation. Job exposure matrix（JEM）was used to assess the exposure to accumulated dust. Uniform Questionnaire was applied toevery subject for collecting the personal information including cigarette smoking, alcoholconsumption, occupational health habit, cognition of occupational hazard, psychological statusand nutrition status. ELISA method was used to determine Serum MMP9/TIMP1 in 174nonsmokers （41 healthy controls and 133 workers）. PCR-RFLP-was used to genotype 519workers by MMP9（C-1562T）、HSP70-1（G190C）、HSP70-2（A1267G） andHSP70-hom（T2437C）.It was found that JEM （Arithmetic mean, AM） in the cases was significantly higher thanthat in the control miners by one-way ANOVA （Ⅰ: 105.4±46.9;Ⅱ: 114.3±40.3;Ⅲ:92.3±34.3; p＜0.01）. Silicosis category has positive correlation to JEM （P＜0.01）. Althougheach index of dust exposure was under the occupational exposure limit （OEL）, the mean dustconcentration of some job categories were over it. At some workplace, the total dustconcentrations reached 30%.The measurement of pulmonary ventilation function showed that each lung function index,including FVC, FEV1.0 and FEV1.0/FVC （measured value and predicted value）, weresignificantly lower in the silicosis group than in the control miners （p＜0.05） and degradedfollowing the increase of JEM category （p＜0.05）. The prevalence of lung dysfunction categorywas trended as follows by Chi-square test: Restricted ventilation dysfunction＞Obstructedventilation dysfunction＞Mixed ventilation dysfunction. The odds ratio （OR） of lungdysfunction in the workers was obtained by Logistic regression analysis and listed as follows,Ⅱ+Ⅲsilicosis （12.557）,Ⅰcategory silicosis （4.863）, BI＜400（2.971）, BI 600～（2.504）,JEM150～（2.390）, BI 400～（2.300）, chronic bronchitis （1.715）.Serum MMP9 in the 64 control miners was significantly higher than that in the 41 healthycontrols by Rank test （p＜0.05）.There was no such relationship in serum TIMP1 （P＞0.05）. Asthe silicosis category increased, the concentrations of serum MMP9 and TIMP1 were also increased, but the ratio of MMP9/TIMP1 was decreased, both of these end points weresignificantly higher in each silicosis category than that in the healthy controls （p＜0.05）.Compared with control miners in the adjusted model considering the JEM category and agecategory, TIMP1 level in I silicosis patients was significantly higher （p＜0.05）, MMP9/TIMP1ratio in theⅡsilicosis patients was significantly higher （p<0.05）, MMP9 and TIMP1 level intheⅢsilicosis patients were significantly higher （p＜0.05）, but MMP9/TIMP1 ratio in theⅢsilicosis patients were significantly lower （p＜0.01）The frequency of HSPT0-2 1267G was significant different in each silicosis category（P=0.046）, moreⅡandⅢpatients carried G allele than that those with I silicosis category andcontrol miners (（P＜0.05）. The frequency of HSP70-hom（T2437C） C/C carriers in theⅡandⅢsilicosis patients was higher than that in control miners andⅠsilicosis patients （8.8% vs2.4%和3%）. Adjusted by JEM category and BI category, silicosis risk in the homozygous major allelecarriers was significantly higher than that in the heterozygous allele carriers and homozygousminor allele carriers（P＜0.05）.The distribution of MMP9-1562C/C carriers and HSP70-2G carriers were marginallydifferent betweenⅡandⅢsilicosis patients andⅠsilicosis and control miners （P=0.087）. Inthe subgroup with JEM more than 150, the frequency of HSP70-hom2437C carriers was alsomarginally different in the silicosis group compared with the control miners （P=0.094）. Nointeraction was found between cigarette smoking and genotypes. HSP70-1（190）、HSP70-2（1267） and HSP70-hom（2437） were highly linked with each other by LDA software.Haplotype analysis by PHASE analysis soft found that G-A-T and G-G-T haplotypes of thesethree SNPs was more frequent in theⅡandⅡcategory silicosis compared to theⅠsilicosisand controls（P＜0.05）.In conclusion, the expression of HSP70-1, HSP70-2,HSP70-hom, HSP27, HO-1 andMMP9/TIMP1 mRNA of HELF could be induced by AMs supernatants exposed to SiO₂,which displayed a clear dose-effect and time-effect relationships. They had differentcharacteristics of induction, which might be caused by the differences of the expression ofregular sequence in the genes. There were phase differences in the expression of differentresponse protein, which could protect the cells in turn. The phase difference of the expressionof MMP9/TIMP1 mRNA indicated MMP9 expression and activity increased in early phase,and ECM degraded. The appearance of TIMP1 inhibited the activity of MMP9, and theMMP9/TIMP imbalance appeared, which prompted collagen deposition. HSP27,PeroxiredoxinⅡand calreticulin had a closed correlation with oxidative-stress induced bySiO₂. In order to prevent silicosis, we not only control the dust concentration in the workplace,but also improve personal protection and decrease the dust exposure at the same time. We alsoneed to enhance occupational health education and health promotion in miners, and identifysusceptible population （HSP70-hom2437C C/C genotype, G-A-T or G-G-T haplotype carriersof HSP70-1, HSP70-2, HSP70-hom）. Struggle for the WHO goal of eliminating silicosis.更多还原