The Related Exploration Of Unfolded Protein Response In Hypoxic Myocardial Cells

Objective:In this study,we established the model of hypoxia myocardium and determine changes of relative contents of GRP78 and XBP-1s mRNA in the reaction of unfolded protein.Then evaluate the unfolded protein response(UPR)activity changes in different periods of hypoxia myocardial cells,which may contribute to reveal the possible mechanism of unfolded protein response(UPR)in cardiac muscle cells.It can provide experimental basis on how to reduce the hypoxia injury and improve the treatment of cyanotic congenital heart disease.Methods:The experiment is divided into three steps:the first:the extraction of myocardial cells from neonatal rat cardiac tissue and cultured;second:the establishment of myocardial hypoxia model;third:the amplification of target gene.The experiments were divided into 6 groups,which were denoted as H0,H6,H12,H24,H48,H72,respectively.Each group cells were exposed to 1%O2,5%CO2 to establish myocardial hypoxia model,then harvest myocardial cells at 0h,6h,12h,24h,48h,72h respectively.Finally collecting the myocardial cell and extract total RNA..Through the application of RT-PCR the expression levels of XBP-1s and GRP78/Bip mRNA were determined,then analyse the relationship between the two groups of data to assess the activity of unfolded protein response(UPR)during different time of hypoxia.Results:In this experiment,With GAPDH as a reference.We regard ratio between optical density of target gene GRP78/Bip and XBP-1s and reference genes as the relative content of target gene.SPSS16.0 software was used for single factor variance analysis.The results show that GRP78/Bip mRNA has an significant difference between H0 and H6,H6 and H24 as well as H24 and H72(p<0.05),which suggest that the expression of GRP78/Bip mRNA are gradually increased with increase of hypoxia time and peaked at 72 hours.while the XBP-1s mRNA has an significant differences in H6,H12,H24 and H48 groups(p<0.05).From the results of XBP-1s mRNA we know that within 6 hours of hypoxia,there was a clear expression,after 12 hours,the amount of expression decreased gradually to the normal level.Conclusion:1.XBP-1s may be one of the important kinases which promote myocardial cell survival at the early stage of hypoxia,but its role in myocardial adaptation to chronic hypoxia is weak,This may be related to the over expression of GRP78/Bip.2.The experimental results also show that the expression of GRP78/Bip mRNA were gradually increased with increase of hypoxia time,which may related to thrombospondin 4.Compared with the XBP-1s,the GRP78/BiP in continuous stress or severe stress may be more meaningful.