The Transcriptomics Study Of Apoptosis Induced By Hydrogen Peroxide In Chicken Myocardial Cells

Apoptosis is a cell death of gene controlling, ordering, and automatic happening, it relates to a series of genes change about the gene activation, expression and regulation. Myocardial cells are terminally differentiated cells, the proliferation ability is very weak, in the late of embryonic period, the number of it almost not increase. Apoptosis and necrosis are the main reason for the decrease of the number of myocardial cells, it is difficult to restore after reducing the number, and can gradually reduce the myocardial cell function, lead to heart failure. In many diseases in chicken, there always companioned with myocardial cell apoptosis, the apoptosis of myocardial cells and even become the initiating factors of some diseases, such as ascites syndrome. Therefore apoptosis is very important in the research of chicken myocardial cells.Transcriptome sequencing can sequence the total RNA in cells at a specific status, including mRNA and non-coding RNA all can be high-throughput sequenced. It is the basis and foundation of study on gene function and structure, through a new generation of high-throughput sequencing, we can obtained nearly all information of all transcriptome in tissue or organ, and it can faithfully reflects the change of expression of all genes within cells.To date, study on the apoptosis of myocardial cells in mammals have been relatively full, in recent years, more and more apoptosis or apoptosis repressor molecules have been found, a large number of apoptosis signal pathway have been explored, the study of its mechanism is more fully. But the study on the apoptosis of myocardial cells in chicken carried out less, many of important molecular and pathway of apoptosis remain unknown, and it is difficult to apply to the results of mammals in chicken. Concern of characteristics of transcriptome sequencing technology, it can be a one-stop solution to these problems. 1.Cultivation and identification of chicken primary myocardial cells and test of its apoptosis inducementTo constructed the model of primary myocardial cells culturing of chicken and obtain stable, high purity of these cells, and investigate the apoptosis inducing conditions,14 d White Leghorn chicken was adopted, separate the ventricular muscle carefully, cut into pieces, 0.05% trypsin digestion, percussion, harvest the cells by centrifugation, inoculated in T25 cell culture flask, after 30 min differential velocity adherent then transfer into 24 hole plate, after 6 h, washing one times, the cultured cells were observed in morphology, immunohistochemical identificated and a PCR of myosin was carried out. The cells were induce by hydrogen peroxide with concentration from 0.1-1.6 mM, monitor the changes of expression of apoptosisgenes. The results showed that trypsin digestion method can obtain high purity of myocardial cells, cells with a phenotype of large triangle, continuous culture led them became multi-morphic. A-actin immunohistochemical staining and PCR results were positive, these cells can steady growth more than 15 days. induced by hydrogen peroxide in 0.2 mM, after 9 h, obviously apoptosis occurred in myocardial cells, gene related apoptosis expressed at a peak value, this concentration can be used for apoptosis induced in experiment. 2.Screening the apoptosis response in chicken myocardial cells by hydrogen peroxide using RNAseqTo investigate these issues and elucidate the pathways and genes that are involved in myocardial cell apoptosis, A primary culture of chicken embryo myocardial cells was maintained and treated with hydrogen peroxide to induce cell apoptosis. RNA extraction via the Trizol protocol and RNA-Seq with technical replicate was carried out. After sequencing, 19,268 known genes and 2,160 novel genes were further investigated. In the experimental groups, 6168 genes showed significant differential expression based on DEseq analysis. Among these genes, 14.5% were upregulated, whereas 17.5% were downregulated.Based on the sequence analysis, a total of 152 pathways were enriched, among them, an majority is the pathway of synthesis and metabolism, but also have some pathways directs related to apoptosis signal pathway. Synthesis and metabolism pathways include: Fattyacid metabolism’, ‘Alanine, aspartate and glutamate metabolism’, ‘Biosynthesis ofunsaturated fatty acids’, ‘Protein processing in endoplasmic reticulum’, the signaling pathway related to apoptosis that be active mainly include: "PPAR signaling pathway", "Adipocytokine signaling pathway", "TGF-beta signaling pathway", "Toll-like receptorsignaling pathway", "MAPK signaling pathway", "signaling pathway P53" and "Wnt signaling pathway". In addition, we found some other pathway were also activated, such as ‘Regulation of actin cytoskeleton’ and ‘Cell cycle’. From the path screening, we found that hydrogen peroxide not only form an apoptosis, but also influenced the function, biosynthesis and metabolism of cells and cell movement, cell cycle. Therefore, hydrogen peroxide induced apoptosis should be a process that very extensive and multiple responses to it.The caspase family and genes that inhibition of apoptosis were used to assessment the cells response to this type of apoptosis, The results showed that the initiation of apoptosis in myocardial cells in the chicken mainly depends on the caspase8 and 9, while the apoptosis effects mainly depend on caspase-3. Inhibition of apoptosis mainly depends on API5 and TRIA1.