The Effect Of HIF-1in Hypoxia Reoxygenation Injury Of Myocardial Cells And The Influence Of Prolyl Hydroxylase SiRA On Its Expression

Objective Using small interfering RNA to intervene the generation of hypoxiainducible factor1alpha (HIF-1α) in cultured rat cardiomyocytes, discuss the effect ofsimulating on myocardial ischemia reperfusion injury, in hypoxia/reoxygenation(hypoxia-reoxygenation, H/R) on the myocardial cell damage repair, influence andchange HIF-1alpha on level downstream target genes, in the clinical treatment andprevention of myocardial infarction in patients suffering from aspects of myocardialischemia reperfusion injury, to provide new ideas for the treatment related.Methods (1)Separate the Primary Cardiomyocytes of SD rat which is1to2daysold, observed the morphological identification of aspects of the separation andpurification of myocardial cells; myocardial cells hypoxia/design and construct ratreoxygenation (H/R) model(2) Design and establish the Hypoxia/Reoxygenation(H/R) model, randomly dividedthe purified primary cardiomyocytes of the SD rat which has been cultured for3to4days into3groups, the specific grouping mode is:①Normal control group (quality control and Control control group);②Hypoxia/reoxygenation (H/R) group;③siRNA transfected rat myocardial cells hypoxia/reoxygenation (P4HA2·siRNAH/R) group;(3) After constructing the oxygen model of hypoxia/reoxygenation myocardial cells,place the cultivation cells under the inverted microscope to count and observe themorphologic changes of cells,including pulsation frequency, survival rate, countingeach cell and each packet in the myocardial cell morphology; detection of myocardial cells proliferation, morphological changes and the survival rate of change (MTSmethod);(4) Construct and identify the small interfering RNA of P4HA2(siRNA), prepare alentiviral vector, transfection into the primary cardiomyocytes cells of the SD rats;(5) Amplified by SYBR Green fluorescence quantitative PCR method, test thecomplex expression level of HIF-1α in each group of Hypoxia/Reoxygenation modelof Cardiomyocytes.Use the chemiluminescence method specific damage marker todetect the content of cardiae Troponin I (cTnI).Detect of downstream target genesVEGF, GLUT-1and HO-1mRNA expression by SYBR Green fluorescencequantitative PCR method. Statistical analysis using SPSS19.0software, usingOne-Way ANOVA analysis and comparison of data among groups, pairwise comparesthe analysis of variance with LSD method.Results (1)After4to6hours the myocardial cells began to grow adhesively. Afterabout15to24hours the myocardial cells began to be fusiform, rhombus or polygon,and emerged the pulsate rhythmic spontaneous.(2)Observed under the inverted microscopes we can see the freshly isolated ratmyocardial cells became spherical.After4to8hours the myocardial cells adherentgrowed fusion irregular oval and fusiform. When about32hours after, most cellsshowed cell clusters that was radiating arrangement, the cytoplasm becomed rich andthe nucleus becomed large. After72hours irregular shape of myocardial cells wereextending pseudopodia, gradually connected into the reticular cell monolayer, thediffraction enhancement and synchronously beating, contraction frequency stably andstrongly. In the fourth day the beat reached to the peak, pulse frequency was about90~120/min,. And in the fifth to sixth days in the state of stable culture conditionsmaintained. From the seventh day of primary cells began to gradually decline, beatingfrequency decreased and the frequency unstably, and then the cells begin to graduallyshrink, cytoplasmic coarse granules and vacuoles, it proved that the cells becomedeath.(3)Establish H/R model successfully(4)Construct and identify of P4HA2small interfering RNA(siRNA), determine the effective targets of siRNA, prepare the lentiviral vector transfection, primary neonatalrat cardiomyocytes, myocardial cells after transfection of H/R model.(5)①About the myocardial cells of each frequency and survival rate with pairwisecompares:The H/R group and normal control group decreased significantly comparedwith the normal group, transfected H/R cells of control group were significantlyincreased (P<0.05);②About the content of each cell of troponin I with pairwise compares:The H/R groupand normal control group increased significantly compared to H/R group, transfectedcells with normal control group were significantly reduced (P<0.05);③About myocardial cells in each group of hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), glucose transporter-1(GLUT-1) andheme oxygenase-1(HO-1) of the expression of mRNA with pairwise compare: TheH/R group and normal control group decreased significantly compared with thenormal group, transfected H/R cells of control group were significantly increased(P<0.05)Conclusion By regulating the downstream target genes, the HIF-1α factor can protectthe hypoxia reoxygenation myocardial cells; after myocardial cells HIF-1α levels inthe use of lentiviral vectors P4HA2siRNA transfection can hypoxia does not appearto increase the overall level is reached, and can effectively regulate a variety ofdownstream target genes were increased, it can be concluded that the use of smallinterfering RNA control Change HIF-1α levels can effectively participate in theprotection previously hypoxia reoxygenation myocardial cells, in order to prevent orreduce the adverse ultimately affect myocardial ischemia and reperfusion suffered.