矽肺大鼠差异表达基因的筛选与验证  

Screen and validation of differentially expressing genes related to silicotic pulmonary fibrosis in rats

作　　者：徐慧蓉[1] 王献华[1] 马小兵[1] 侯文娜[1] 朱兰[1] 向聚才[1] 孙瑞军[1] 

机构地区：[1]河北联合大学基础医学院病理学教研室,唐山063000

出　　处：《中华劳动卫生职业病杂志》2012年第1期45-51,共7页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：河北省自然科学基金资助项目（C2008001007）;河北省科学技术研究与发展计划项目（09276192D）

摘　　要：目的 筛选矽肺大鼠肺组织与正常大鼠肺组织的差异表达基因,并对差异表达基因基质金属蛋白每-12（MMP-12）和组织蛋白酶E（Cathepsin E）进行鉴定,探讨其在矽肺发病中的作用.方法 将健康SD大鼠随机分为模型组（24只）和对照组（6只）,采用非气管暴露法建立雄性SD大鼠矽肺模型,HE染色和VG染色进行肺组织病理观察.对染尘60d的大鼠肺组织,应用基因芯片筛选大鼠肺组织中的差异表达基因.选择明显上调的MMP-12和Cathepsin E基因,用反转录聚合酶链式反应（RT-PCR）、免疫组织化学法及蛋白质印迹（Western blot）法进行鉴定.结果 从26 962个基因中共筛选出模型组和对照组的差异表达基因338个,其中上调基因267个,下调基因71个.经RT-PCR验证,染尘后30、60、90d时,模型组大鼠肺组织中MMP-12 mRNA表达的吸光度值分别为对照组的4.306、5.338、6.713倍,Cathepsin E分别为1.434、2.974、3.889倍.经免疫组织化学法验证,染尘后30、60、90 d时,模型组大鼠肺组织中MMP-12蛋白表达的平均吸光度值分别为对照组的1.435、1.746、2.069倍,Cathepsin E分别为1.372、1.663、2.103倍.经Western blot法验证,染尘后30、60、90d时,模型组大鼠肺组织中MMP-12蛋白表达的平均吸光度值分别为对照组的1.214、1.531、1.959倍,Cathepsin E分别为1.262、1.828、1.907倍.与对照组相比,模型组大鼠肺组织中MMP-12和Cathepsin E mRNA和蛋白表达量明显上调,差异均有统计学意义（P＜0.05）.结论 应用基因芯片筛选出矽肺大鼠肺组织差异表达基因并得以验证,MMP-12和Cathepsin E可能通过降解肺泡壁基底膜及参与免疫应答等参与矽肺的发生发展过程.Objective To screen the differentially expressing genes between silicotic lung tissue and normal lung tissue,to identify the differentially expressing genes of matrix metalloproteinase-12（MMP-12）and Cathepsin E and to explore the roles of those genes in silicosis development.Methods Thirty male SD rats were divided randomly into two groups： control group（6 rats）and exposure group（24 rats）which was exposed to SiO2 by intra-tracheal perfusion.On the 30 th,60 th and 90 th days after exposure,8 rats in model group and 2 rats in control group were executed and the lung tissues were obtained.The morphologic changes of lung tissues were observed with HE staining and VG staining under a light microscope.The gene microarrays were used to identify differentially expressing genes of lung tissues in rats exposed to SiO2 for 60 days.Two significantly up-regulated genes,MMP-12 and Cathepsin E,were validated using RT-PCR,immunohistochemistry and Western Blot assay.Results A total of 338 differentially expressing genes were identified from the 26 962genes between silicotic rats and normal rats,including 267 up-regulated genes and 71 down-regulated genes.The results of RT-PCR showed that in the lung tissues of exposure group on the 30 th,60 th and 90 th days,the mRNA expression levels of MMP-12 were 4.306,5.338,6.713 times higher than those in the control group,the mRNA expression levels of Cathepsin E were 1.434,2.974,3.889 times higher than those in the control group,respectively.The results of immunohistochemical showed that in the lung tissues of exposure group on the 30 th,60 th and 90 th days,the mRNA expression levels of MMP-12 were 1.435,1.746,2.069 times higher than those in the control group,the mRNA expression levels of Cathepsin E were 1.372,1.663,2.103 times higher than those in the control group,respectively.The results of immunohistochemical showed that in the lung tissues of exposure group on the 30 th,60 th and 90 th days,the expression levels of MMP-12 protein were 1.214,1.531,1.959 times higher th

关 键 词：矽肺 基因表达 基质金属蛋白酶 组织蛋白酶E 

分 类 号：R135.2[医药卫生—劳动卫生]