Study On The Different Gene Expression Associated With PGE2of Chondrocyte Treated By The Celecoxib Under Inflammatory Situation By The Microarray

Objective To compare and find out the differential expression of gene between the chondrocyte treated by COX-2selective inhibitor and non-treated by COX-2selective inhibitor under inflammation by Affymetrix microarray, indentifying the difference of the genes expression associated with the COX-2and PGE2, providing gene datas for the effect of celecoxib. The proteins and mRNA which encoded COX-2and PGE2were verified simultaneously by the RT-PCR and ELISA to confirm the accuracy of the miroarray.Methods The cartilages coming from three cases, including two specimens came from TKA surgery for OA and one case came from normal specimens of knee cartilage.The condrocyte from these cartilage were cultured and divided three groups, including the negative control, positive control (stimulated by IL-1β) and intervention group (celecoxib+IL-1β).Under the hypothermic situation, then the total RNA was extracted and detected. Hybridization with Affymetrix Human microarray was performed after the RNA was labeled with fluorescent probe. The image scanning and data analysis were performed, comparing the data between negative group and positive group, the positive and the intervention group, identifying different expression associated with the COX-2and PGE2genes. Finally, the partial results of hybridization associated with the COX-2and PGE2genes were validated by Real-time PCR and ELISA.Results First part:1)212differential expression genes were screened between the negative group and positive group by the microarrays in the four cases. Theses gene transcripts associated with the biosynthesis and degeneration of ECM、immune or inflammation、proliferation and apoptosis. Differential expression genes associated with COX-2/PGE2were upregulated, including PTGS2(encoding COX-2)、PTGES (encoding PGE synthase). PTGER4(encoding PGE2receptor)、MMP13(cncoding MMP13), the PTGS1(encoding COX-1) was downregulated.2)156differential expression genes were screened between the positive group and intervention group by the microarrays in the four cases. Differential expression genes associated with COX-2/PGE2:PTGS2(encoding COX-2)、PTGES (encoding PGE synthase)、 PTGER4、MMP13were downregulated; PTGS1(encoding COX-1)、was upregulated. Second part:It was confirmed that IL-1βincreased the COX-2、 PGE2、 EP4expression, consequently promoted the expression associated with the degeneration of the cartilage by RT-PCR and ELISA. Celecoxib decreased the expression of COX-2、PGE2、EP4and MMP, but did not change the expression of EP2(the PGE2receptor).Conclusion IL-1βincreased the COX-2、PGE2and EP4expression, consequently promoted the degeneration of the cartilage. Celecoxib inhibited the activity of COX-2meanwhile decreased the expression of COX-2、PGE2、EP4and MMP, prevented the degeneration of cartilage from inflammation.