Primary Study On Gene Expression Profile Of Cytokines In Myocardium And PBMC During Cardiopulmonary Bypass

Objective: To determine the application values of gene chip technique in cardiovascular surgery clinic and study. Microarray for gene expression profiles was used to screen out the differentially expressed genes during CPB in myocardium and PBMC. Semiquantitative reverse-transcription polymerase chain reactions were used to confirm the selected cDNA microarray results. The candidate genes and its functions were primarily analyzed and forecasted. By doing these, we hoped that we could find out some clues in inflammatory response during CPB and common functional cytokines in inflammation. Furthermore, we hoped that we could explore the possible site to modulate inflammation and give some hints in pharmacal study as well as clinical intervention. We also hoped it could be a groundwork during the following study and clinical work.Methods: The inpatients underwent open heart surgery under CPB were selected. Right atrium were collected just before onset and termination of CPB. The specimens were stored immediately in liquid nitrogen until use. The patients' oxygenated bloods were drawn immediately before onset and termination of CPB. PBMC were obtained from heparinised blood by Ficoll gradient centrifugation. The total RNA was collected using TRIzol Reagent. The following steps were performed according to the manual of BD AtlasTM cDNA Expression Arrays: cDNA probe synthesis; cDNA probe column chromatography; hybridizing cDNA probes to the BD Atlas?Array and cDNA probes stripping. Microarrays were exposured to phosphorimager and scanned. The images were preceded and analyzed using AtlasImage1.01aAbstractsoftware. The results were analyzed according to BD Atlas?Gene Lists Version 5.0. The candidate genes were corroborated by semiquantitative RT-PCR in clinic. The candidate genes and its protein functions were primarily analyzed by bioinformatics.Results:1 . For the first time, gene chip technique was successfully used in CPB study. cDNA microarray for gene expression profiles could screen out differential gene expression during CPB in a parallel, fast and large scale manner. The results of cDNA microarray were not completely coincident with the results of RT-PCR. Moreover, it might be reverse. BD Atlas?cDNA Expression Arrays could be used at least three times. But the reuse resulted in higher background and influenced the result. However, the results were satisfying within three times.2. The gene expression profiles of cytokines in myocardium during CPB were screened out for the first time. Cytokines in myocardium during CPB presented differential expression. IL-6, IFN- Y and WntSa were downregulated according to cDNA microarray but upregulated by RT-PCR. IL-13 and G1P3 were upregulated according to cDNA microarray but of no significant differences by RT-PCR. TNFRSF1B, P1GF and MFNG were all upregulated in cDNA microarray and RT-PCR. The number and degree of differentially expressed genes of cytokines in myocardium were both more serious than that of PBMC.3. The gene expression profiles of cytokines in PBMC during CPB were screened out for the first time. Cytokines in PBMC during CPB presented differential expression. IL-6 was downregulated and WntSa upregulated according to cDNA microarray but no significant differences were found in-10-Abstractboth by RT-PCR.4. The gene expression profile before CPB in different tissues was distinct. The significant differences were IL-6, WntSa and discoidin domain receptor family, member 2.5. The gene expression profile after CPB in different tissues was distinct. The significant differences were P1GF and WntSa.6. IL-6 and IFN- y which have relations with inflammation and immuneregulation were upregulated after CPB in myocardium. It was the first time to find IFN- y upregulated.7. For the first time, TNFRSF1B known as the receptor of TNF- a was found out upregulated during CPB. TNF- a is essential in inducing inflammation. TNF- a has much more effects on myo...