Searching For Altered Altered Gene Expression Of Homocysteine Treated ECV-304 Cells By CDNA Microarry

Background and Objective: Coronary Heart Disease(CHD) , which is also named Ischeraic Heart Disease, is caused mainly by atherosclerosis and exists as a kind of commonly encountered disease in the clinic that threatens severely the health. The morbidity rate of CHD has begun to show a trend of rising and low-age occurrence from 1980' s. According to estimation of the World Health Organization (WHO) , China, as well as the other developing countries, will meet their CHD peak in 2002. One of the effective measures to lowen morbidity risk is to eliminate , as actively as possible, the factors that tend to result in CHD, such as hyperlipidemia, hypertension, smoking, diabetes, fatness, etc. Furthermore, the latest research indicates that the hyperhomocysteinaemia(HH(e)) is also a independent risk factor of CHD. The theory of "response-to-injury hypothesis of atherosclerosis" proposed by Ross emphasizes that the damage of endothelial structure and function is a crucial mechanism initiating the pathogenesis of atherosclerosis. In addition, the present researches prove that high concentration HCY (homocysteine) leads to structural and functionaldamage of the vessel endothelial cells because its toxicity can have influence on endothecium, endothelial-dependent diastolic function and endothelial adherent function, etc. Howerver, the underlying molecular mechanisms for HCY-induced endothelial damage is not fully understood. The classic RNA methodologies such as Northern blot analysis can only study one gene at a time in higher price and lower efficiency, while the jumped-up DNA microarray technology provides a high-throughput tool for an unbiased screening of differentially displayed genes. In this experiment, changes of gene expression in ECV-304 cells induced by high concentrations HCY are examined though the DNA microarray method, in order to study the molecular mechanism of endothelial damage and atherosclerosis caused by HCY.Methods: ECV-304 cells were planted in DMEM medium including 20% fetal bovine serum (FBS) and placed in C02 cultivable box (37癈, 5% C02, and saturated humidity). The medium was changed every 48 hours. When cells density reached to 80%, the medium was replaced with serum-free medium . At the same time , all cells were divided into two groups. One was control group, treated with 10umol/L HCY ; the other was test group, treated with 500 mol/L HCY . Both were placed in C02 cultivable box and cultured for another 24 hours. Then, total RNA was extracted from collected ECV-304 cells of the two groups and cDNA probes were made respectively with fluorescent label dyes by reverse transcription method. That is, the control group was labeled by Cy3-dCTP and the test group by Cy5-dCTP. After pre-hybridization, hybridization and washing, the hybridizing signals was gathered by scanning with Scan Array 4000. Process above was repeated and two pieces of gene chips were attained at last.Results: In both chips there were about 3977 genes scanned each, of which 155 and 186 genes respectively showed differential expression. And 52 genes were the common differentially expressed ones in gene chip 1 andgene chip 2. 47 genes were up-regulated and 5 genes were down-regulated. The up-regulated genes included:(1)genes related to protein synthesis: Genebank_ID: NM₀01961 (EEF2 mRNA) ; NM₀00635 (RFX2 mRNA); NM₀02695(POLR2E mRNA); NM₀04184(WARS mRNA); (2)Cyclins: NM₀01950(E2F4 mRNA); NM₀00302 (PLOD mRNA); NM₀05777 (RBM6 mRNA); NM₀31846 (MAP2 mRNA);(3) genes related to DNA binding, transcript and transcript factor.-NMJH4233 (UBTFmRNA); NM₀12225(NUBP2mRNA); NM₀01521 (GTF3C2 mRNA); NM₀05839 (SRRM1 mRNA); NM₀01455 (FOXOSAmRNA); (4) genes related to ion channels and transporters: NM₀02996 (CX3C mRNA); NM₀30878; NM₀04519(KCNQ2 mRNA); NM₀03562 (SLC25A11 mRNA); NM₀05628 (SLC1A5 mRNA); (5) genes related to cytoskeleton and cell movement:NM₀03827; NM₀01102(ACTN1 mRNA); NM₀06417 (IFI44 mRNA; NM₀02385 (MBP mRNA); NM₀04475(FLOT2 mRNA); (6)genes related to cell receptors: NM₀06801(KDELR1 mRNA); NM₀0...