基因芯片技术研究Z24对人HepG_2细胞基因表达的影响

Effects of Z24 on the gene expression profiles of human HepG_2 cells with DNA microarray

目的:利用芯片技术观察Z24对人HepG2细胞基因表达的影响,以探讨其毒性作用的分子机制。方法:不同浓度处理培养的HepG2细胞,H-E染色观察细胞形态;抽提细胞RNA,利用荧光标记ddUTP逆转录制备cDNA探针,与毒理表达谱基因芯片杂交,并对Cy3,Cy5荧光信号做扫描分析。结果:Z24处理实验组细胞呈现凋亡形态;芯片扫描显示在IC20浓度情况下,Z24作用HepG2细胞有15个基因发生显著的变化,其中细胞凋亡通路有关的基因TNFRSF5发生明显上调,与肝功能有关的基因(AKR1C2和INSIG1)发生明显的下调。随Z24浓度的增加,发生改变的基因数增加到244个,其中109个基因发生明显的上调,135个基因发生明显的下调,下调的基因多与细胞增殖调控功能、氨基酸、能量和脂肪代谢有关;而细胞凋亡通路中几个重要关键蛋白(DFFB,CASP6,DR4)的基因明显上调,并且与肝脏有关的基因(INSIG1,PHKA2,HPX)也发生了明显的改变。结论:Z24可以诱导HepG2细胞凋亡,并可能是其产生毒性的重要机制之一;毒性作用的靶器官可能是肝脏。

Objective:To explore the effects of Z24 on the gene expression profiles of human HepG2 cells with microarray technique and further investigate the molecular mechanism of Z24 toxicology . Methods: In exploration for the gene expression of HepG2, after treated with Z24 in IC20 and IC50 for 24h,the HepG2 cells were observed under microscope with H - E staining. To prepare the probes, mRNA from both control and treated cells were isolated and purified, then reversely transcribed to cD-NA with the incorporation of fluorescent-labeled ddUTP.The probes were hybridized with cDNA microarray representing the 1 153 genes originated from human. The fluorescent signals of Cy3 and Cy5 were scanned and analyzed. Results: The HepG2 cells showed a typical feature of apoptosis after treatment with Z24. The gene expression profiles were also changed greatly. There were 15 genes in IC20 in which the key gene TNFRSF5 involved in apoptosis signal transduction was up-regulated and liver functional related genes like AKR1C2 and INSIGl were down-regulated. Among the 244 gene changed in IC50 ,109 of them were up-regulated and 135 were down-regulated. They were functionally related to cell proliferation cycle, replication, and metabolism and so on. Several apoptosis associated genes were detected in the up-regulated genes, encoding the key protein involved in apoptosis signal transduction, such as DFFB,CASP6,DR4. Liver function related genes like INSIG1, PHKA2, and HPX were also changed. Conclusion: The Z24 can initiate the apoptosis process of HepG2 cells and the latter may affect the toxicology in liver.