摘要
[目的]研究莪术醇对肝星状细胞(HSC)T6细胞基因表达的影响。[方法]从基因库中查询50种肝纤维化相关基因的mRNA序列,用寡核苷酸探针设计软件设计探针,在PE8909DNA合成仪上合成寡核苷酸,用OGR04点样仪及醛基化玻片制备成基因芯片。以莪术醇不同浓度含药培养液培养HSC T6细胞,根据细胞毒性实验,确定细胞存活率在50%以上的药物浓度作为实验所用浓度,每组设空白对照,分别按1、6、12、24h4个时间段收集细胞。按操作步骤提取细胞总RNA,经反转录荧光标记,杂交和洗涤,用Genepix4000B扫描仪扫描芯片,ImaGene4.2软件进行数据分析和归一化处理,使用看家基因和阳性对照对Cy3和Cy5扫描结果进行校正。[结果]莪术醇1.5625μg/ml作用HSC T6细胞12h,可使基因转化生长因子β1、细胞色素P450a表达下调2.3、2.1倍。[结论]从分子水平揭示了莪术有效成分莪术醇的抗肝纤维化机制。
Objective] To investigate the effects of component of curcumol on the gene expression in cultured activated hepatic satellite cells,and explore the molecular mechanism of anti-hepatic fibrosis for curcumol at gene network level.[Methods] The mRNA sequences of 50 liver fibrosis-related genes were consulted from the gene bank. Oligonucleotide probes were designed by software, oligonucleotides synthesized by PE8909DNA synthesizing instrument, and oligonucleotide microarray made with OGR-04 droping instrument and aldehyded glass chip. Cultured HSC-T6 cells were treated with different concentrations of curcumol. According to the experiment of cells toxicity,the appropriate concentration of medicine, in which cells livability was higher than 50 %, was taken as experimental concentrations. At the same time, the blank comparison was set up in each team,and cells at 4 points of 1 h, 6 h, 12 h, 24 h were gathered respectively. Total RNA was distilled with Trizol reagent according to the operation process,cDNAs labeled from total RNA with Cy3-dUTP and Cy5-dUTP. These labeled cDNAs were hybrided to an oligonucleotide microarray,then the oligonucleotide microarray was washed a few times. Afterwards, the gene microarray was scanned by scanner Genepix 4000B,and the difference of gene expression of HSC-T6 cells was analysed with ImaGene 4.2 software.[Results] After HSC-T6 cells were cultured in medium with 1.562 5 μg/ml of curcumol for 12 h, gene expression of TGFβ_1 and P_(450)a were down-regulated 2.3 and 2.1 bolds,respectively.[Conclusion]These results show not only the possible molecular mechanism of anti-hepatic fibrosis of curcumol,but also the possibility of TCM research by DNA microarray.
出处
《中国中西医结合消化杂志》
CAS
2005年第3期144-147,共4页
Chinese Journal of Integrated Traditional and Western Medicine on Digestion
基金
广东省中医药管理局课题(102135)
深圳市科技局课题(200204187)
关键词
基因芯片
莪术醇
肝纤维化
肝星状细胞
DNA microarray
curcumol
hepatic fibrosis
hepatic stellate cells
