抑制消减杂交技术在筛选矽肺大鼠差异表达基因中的应用  

Application of suppression subtractive hybridization in screening of differentially expressed genes related to silicotic rats

作　　者：侯文娜[1] 马小兵[1] 徐慧蓉[2] 陈阳[3] 朱兰[1] 伊雪[1] 

机构地区：[1]河北联合大学基础医学院,河北唐山063000 [2]淄博市中心医院,山东淄博255036 [3]河北联合大学生命科学学院,河北唐山063000

出　　处：《工业卫生与职业病》2014年第6期427-432,共6页Industrial Health and Occupational Diseases

基　　金：河北省科学技术研究与发展计划项目(09276192D)

摘　　要：目的对正常大鼠肺组织与矽肺大鼠肺组织之间基因表达差异进行比较,筛选与矽肺相关的差异表达基因片段。方法 30只SD大鼠随机分为2个组,对照组6只,实验组24只。实验组大鼠采用非气管暴露法染尘制作矽肺大鼠模型,对照组灌注生理盐水,利用HE切片确定建立模型成功;抑制消减杂交技术构建cDNA双向消减文库;菌液RCR技术初步筛选cDNA消减文库,将获得的阳性克隆进行测序、同源性比对及生物信息学分析。结果扩增消减cDNA文库获得400多个白色阳性克隆,随机挑取252个白色克隆用PCR进行扩增,95%的克隆中均有100-1 500bp的插入片段,经测序,得到具有差异表达的基因175个。经生物信息学分析表明,筛选出的已知差异表达基因与众多生物学过程和分子功能相关,包括物质代谢、物质转运、细胞增殖、细胞凋亡、细胞黏附、信号转导等,其中许多差异表达基因与矽肺的关系尚未被报道。结论利用抑制消减杂交技术可以建立高质量的矽肺正、反向差异消减cDNA文库,筛选与矽肺相关的已知和未知基因,为寻找矽肺诊断和预后的检查指标和筛选治疗靶点提供有用的信息。Objective To compare gene express difference between in the lung tissue of silicotic rats and in the normal ones so as to screen the differentially expressed genes in the silicotic rats.Methods 30 SD rats were divided randomly into two groups：6in control group and 24 in exposure group which was exposed to SiO2 by intra-tracheal perfusion.The morphologic changes of the lung tissue of the rats were observed with HE staining.A library which contains the differently expressed subtracted cDNAs between normal lung tissue and silicotic lung tissue was constructed with suppression subtractive hybridization.The colony PCR method was used to screen the subtracted cDNA library with forward-and reverse-subtracted cDNA library.The differentially expressed cDNA fragments in silicotic rats was sequenced and analyzed in Genebank with Blast search.Results The amplified library contained more than 400 positive clones.Random analysis of 252 clones with colony PCR method showed that 95% of the clones contained the 100~1 500 bp inserts,175 inserts might be the cDNA fragments of differentially expressed genes in silicotic rats.Bioinformatics analysis showed that most function-known genes had important roles in the structure and movement of cell or extracellular matrix,material transportation,apoptosis,cell adhesion,regulation of metabolism,signal transduction and so on,in which,many genes related to lung fibrosis have not been previously reported.Conclusions Using of suppression subtractive hybridization can create high quality differently expressed subtracted cDNA library for the screening of known and unknown gene ralated to silicosis.Systemic analysis of gene expression profiles may play major roles in the understanding of silicosis development and offer the opportunity for searching marker of diagnosis and prognosis as well as provide clues for targeting therapy.

关 键 词：矽肺 差异表达基因 抑制消减杂交 CDNA消减文库 

分 类 号：R364.3[医药卫生—病理学]