摘要
目的:研究莪术4种有效成分(莪术油、莪术醇、β榄香烯、姜黄素)对肝星状细胞T6(HSCT6)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。以秋水仙碱、莪术油、莪术醇、β榄香烯、姜黄素不同浓度含药培养液培养HSCT6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,每组设空白对照组,分别按1、6、12和24h4个时间段收集细胞。按操作步骤提取细胞总RNA,经逆转录荧光标记、杂交和洗涤,用Genepix4000B扫描仪扫描芯片和ImaGene4.2软件进行数据分析并归一化处理,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:秋水仙碱6.25μg/ml作用HSCT6细胞12h,可使基质金属蛋白酶组织抑制剂1(TIMP1)表达下调220%;莪术油78.125μg/ml作用HSCT6细胞24h,可使基因TIMP2、白细胞介素6表达分别下调230%和220%;莪术醇1.5625μg/ml作用HSCT6细胞12h,可使转化生长因子β1、P450a基因表达下调230%和210%。结论:本研究从分子水平揭示了莪术有效成分莪术油、莪术醇的抗肝纤维化机制。
Objective: To investigate the effects of four different components 〔curcuma aramatica oil(CAO),curcumol, βecement and curcumin〕 of Ezhu(莪术) on the gene expression of hepatic stellate cellsT6(HSCT6). Methods: The mRNA sequences of 50 liver fibrosisrelated genes were looked up from a gene bank and oligonucleotide probes were designed by software of designing oligonucleotide probe. Oligonucleotides were synthesized PE8909DNA synthesizing instrument. OGR04 dropping instrument and aldehyded glass chip were used for the preparation of oligonucleotide microarray. Cultured HSCT6 cells were treated with different concentrations of colchicines, CAO, curcumol, βecement and curcumin. According to the experiment of cell toxicity, the appropriate concentrations of medicine used were determined by the survivor rate more than 50%. The blank control of each group was set up and the cells were collected at 1, 6, 12 and 24 hours respectively. According to Trizol manua, Trizol was applied in the extraction of the total RNA. The labeled cDNAs were prepared from the total RNA by the reverse transcription fluorescein labeling method, and in the preparatory process Cy3dUTP and Cy5dUTP were applied. These labeled cDNAs hybridized with the oligonucleotide microarray was rinsed for a few times. Afterwards, The gene microarray was scanned by scanner Genepix 4000B and the different gene expressions of HSCT6 cells were analyzed with Ima Gene 4.2 software. Results: After HSCT6 cells were cultured in a medium with 6.25 μg/ml of colchicines for 12 hours, gene expression of tissue inhibitor of metalloprotenase 1(TIMP1) was downregulated by 220%, and in a medium with CAO 78.125 μg/ml for 24 hours, gene experessions of TIMP2 and interleukin6(IL6) were downregulated by 230% and 220%,respectively. After HSCT6 cells were cultured in a medium with curcumol 1.562 5 μg/ml for 12 hours, the gene expressions of transforming growth factorβ_1(TGFβ_1)and P450a were downregulated by 230% and 210%, respectively. Conclusion: These results show the
出处
《中国中西医结合急救杂志》
CAS
2005年第3期135-139,i001,共6页
Chinese Journal of Integrated Traditional and Western Medicine in Intensive and Critical Care
基金
广东省中医药管理局课题资助(102135)
深圳市科技局资助课题(200204187)
关键词
基因芯片
莪术
肝纤维化
肝星状细胞
DNA microarray
Ezhu
hepatic fibrosis
hepatic stellate cells
