摘要
目的研究经二氧化硅(Si O_2)染毒的肺成纤维细胞(LF)转分化前后全基因组蛋白H3赖氨酸4(H3K4)三甲基化(met3)水平的改变。方法原代培养大鼠肺泡巨噬细胞(AM)和LF,采用transwell共培养,建立共培养模型,用100 mg·L^(-1)游离Si O_2粉尘染毒AM 24 h,收集LF细胞,免疫组织化学法对转分化后的LF进行表型鉴定。采用染色质免疫共沉淀联合芯片技术(Ch IP-chip)对LF全基因组蛋白H3K4met3进行高通量筛选,采用染色质免疫共沉淀-实时荧光定量聚合酶联反应技术(Ch IP-q PCR)验证芯片结果,采用定量逆转录聚合酶联反应技术(q RT-PCR)检测H3K4出现显著差异的m RNA表达水平。结果通过对经Si O_2染毒前后LF全基因组蛋白H3K4met3水平的对比,共筛选出1815个基因存在H3K4显著性差异(Cy3/Cy5比值>2.0或<0.5,Nimble Scan V2.5软件),其中518个基因出现H3K4met3程度增高,1297个基因H3K4met3程度降低。随机挑选2个差异表达基因nfib和kpna3,以Ch IP-q PCR和q RT-PCR方法验证,取得与Cp G岛芯片一致的结果。结论经Si O_2染毒的LF转分化前后全基因组蛋白H3K4met3水平存在显著改变。Ch IP-chip技术有利于进一步揭示矽肺纤维化发生的分子机制,有助于发现新的蛋白标志物和基因干预靶点。
OBJECTIVE To analyze trimethylation of genome-wide histone H3 lysine 4（H3K4met3）induced by silicon dioxide（Si O_2）through chromatin immunoprecipitation linked to microarrays（Ch IPchip）in lung fibroblast（LF）of rats. METHODS A primary co-culture model of rat alveolar macrophages（AM）and LF in vitro. AM were exposed to 100 mg·L（-1）free Si O_2 for 24 h,before LF were collected and the phenotype of LF was determined after transdifferentiation by immunohistochemistry. Ch IP- chip was used to profile the variations of trimethylation in H3K4 of lung fibroblasts in Cp G island regions. Ch IPq PCR was used to validate the microarray results. The m RNA expression of nfib and kpna3 was analyzed by q RT- PCR. RESULTS Totally 1815（518 increased and 1297 decreased） genes of H3K4met3 displayed significant differences in Si O_2100 mg·L（-1）group compared with control group（Cy3/Cy5 value2.0 or 0.5,Nimble Scan V2.5 software）. The results of Ch IP-q PCR were quite consistent with those of microarray. CONCLUSION There are significant differences in methylation of genomewide H3K4 between Si O_2100 mg·L（-1）group and control group. These novel candidate genes may become potential biomarkers or new interfered targets.
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2016年第7期728-735,共8页
Chinese Journal of Pharmacology and Toxicology
基金
国家自然科学基金(81102109)
国家自然科学基金(81472954)~~
