Investigating Effects Of Different Components Of EZHU On Gene Expression Of Cultured HSC-T6 Cells With DNA Microarray

Objective: We constructed an oligonucleotide microarray with 50 probes representing 50 hepatic fibrosis-associated genes.We substituted the HSC-T6 cells for original hepatic stellate cells, aimed to investigate the effects of 4 different components of EZHU ( curcuma aramatica oil, curcumol, p-ecement, Curcumin ) on the gene expression in cultured activated hepatic stellate cells, and explored the molecular mechanism of anti-hepatic fibrosis for EZHU at gene network level.Methods: We looked up the mRNA sequence of 50 liver fibrosis-related genes such as TGF β1, PDGF, IL-6, IL-10, HGF, ICAM-1 MIP-2, TGF β R I , TGF β R II , PDGFR α , PDGFR β , TNF α R1, MMP2, TIMP-1 from the gene bank, designed oligonucleotide probes by software of designing oligonucleotide probe.We synthesized oligonucleotides in PE8909 DNA synthesizing instrument,and maked out oligonucleotide microarray with OGR-04 droping instrument and aldehyded glass chip. Cultured HSC-T6 cells were treated with different concentrations of medicines ( colchicine, curcuma aramatica oil, curcumol, β-ecement, curcumin ) . According to the experiment of cells toxicity,we took the appropriate concentrations of medicines whose cells livability was higher than 50% as experiment concentrations. We setted up the blank comparison each team in the same time,and gathered the cells at 4 point of Ih, 6h, 12h, 24h, respectively.We distilled total RNA with Trizol reagent according to the operation process, then labeled cDNAs from total RNA with cy3-dUTP and cy5-dUTP.These labeled cDNAs were hybrided to an oligonucleotide microarray,then we washed the oligonucleotide microarray a few times. Afterwards, we scanned the gene microarray by scanner Genepix 4000B,and analysed the difference of gene expression of HSC-T6 cells with ImaGene 4.2 software.Results: After HSC-T6 cells were cultured in medium with 6.25g/ml of colchicine for 12h, gene expression of TIMP-1 was down-regulated 2.2 bolds,TIMP-2 and IL-6 were down-regulated 2.3, 2.0 bolds, respectively, after HSC-T6 cells were cultured in medium with curcuma aramatica oil 78.125g/ml for 24h. Meantime, TGF β1 and p450a were down-regulated 2.3 and 2.1 bolds, respectively, after HSC-T6 cells were cultured in medium with Curcumol 1.5625g/ml for 12h.Conclusions: These results not only show the possible molecular mechanism of anti-hepatic fibrosis of CAO and Curcumol, which are components of EZHU, but also the possibility of TCM research by DNA microarray.