邻苯二甲酸二酯诱导果蝇脑组织基因差异表达谱的研究

Study on the Gene Expressed Profile of Di(2-ethylhexyl) Phthalate-induced Brain Tissue in Drosophila melanogaster

[目的]获得邻苯二甲酸二(2- 乙基己基)酯[di(2 -ethylhexyl)phthalate ,DEHP]诱导果蝇脑组织的基因差异表达谱。[方法]设喂饲0 .2 %DEHP浓度的果蝇为实验组,乳化剂为对照组,2周后断头进行总RNA抽提,DNaseΙ消化去除DNA污染;用OligotexmRNAMiNiKit(QIAGEN)得到纯的mRNA ;合成双链cDNA ;采用体外转录的方法获得大量cRNA与Affymetrix的果蝇表达谱芯片进行杂交,计算机扫描处理数据。[结果]差异表达的基因片段共计3 5个:①上调的基因有2 0个,包括CYP6A2、CYP6A8、CYP6W1、CYP4P1、UGT3 6Bb、GST、羧酸酯酶、转甲基酶、氧化还原酶、EST10、JHEH1、KEAP1、CG690 8、CG1942、CG10 5 60、CG115 2 9、CG10 5 5 3、CG12 83、CG12 5 0 5等;②下调的基因有15个,包括MST5 7Da、MST5 7Db、ACP95EF、OS E、OS C、PBPRP3、PBPRP1、A10、CG1862 8、CG113 91、CG182 84、CG842 4、CG5 2 90、CG13 947、CG15 2 63等。差异表达基因中大多数与信号传导、细胞代谢、生长、发育、组织分化及转录因子等功能相关,尚有一些基因功能未知。[结论]运用高通量基因芯片技术获得DEHP诱导果蝇脑组织的基因差异表达谱,为从分子水平系统研究DEHP的毒性机制提供有益的线索。

Objective] To obtain the gene expression profile of DEHP-induced brain tissue in Drosophila melanogaster. [Methods] Drosophila melanogaster were treated with 0.2% DEHP as a test group while those with saccharose as a control one.The total RNAs were isolated from Drosophila melanogaster head after 0.2%DEHP and saccharose exposure for two weeks,digested with DNase Ι to get pure RNAs. Oligotex mRNA MiNi Kit(QIAGEN) was used for isolation of poly A+ mRNA from total RNAs. dsDNAs were synthezised. IVT with the biotin-labeled was used to prepare for cRNAs,then hybrided with the Affymetrix Oligo chip. The data were scaned and analyzed by computer. [Results] 35 genes fragments were shown in differential expression profile with 20 upregulated genes,including CYP6A2,CYP6A8,CYP6W1,CYP4P1,UGT36Bb,GST,Carboxylesterase,Glycine N-methyltransferase,Oxidoreductase,EST10,JHEH1,KEAP1,CG6908,CG1942,CG10560,CG11529,CG10553,CG1283,CG12505 and 15 downregulated genes,including MST57Da,MST57Db,ACP95EF,OS-E,OS-C,PBPRP3,PBPRP1,A10,CG18628,CG11391,CG18284,CG8424,CG5290,CG13947,CG15263. These genes involved in the functions of signal transduction,cell metabolism,growth,transcription factor. [Conclusion] The data have provided useful clues to studying the toxicological mechanisms of DEHP at molecular level.