The Mechanism Of Hypoxia On Rat Myocardial Cell Damage

Objcective This study investigates hypoxia on rat myocardial cell damage and cause damage mechanism,for the clinical treatment of the disease such as heart failure data accumulation theory.Method(1)Experimental design and grouping: in rat myocardial cell lines(H9C2)as material,design the experimental group and control group,experimental conditions for 1% O2 + 5% CO2 + 1% N2,37 ? under the condition of three gas incubator of hypoxia training;The control conditions for Air 37 ?,5% CO2,95% CO2 incubator normal training.Respectively for 3 h,6 h,12 h,24 h,48 h time based,the indicators of observation cell.(2)Observation of cell growth and vitality: segmented out using trypan blue staining,to its using automatic cell count meter for cell count and cell growth curve drawing;Using the method of ELISA using CCK 8 OD value of the kits each time point to determine the cell vitality.Using cell real-time viewer(Cellasic)at different time points,the change of the cell growth and continuous observation of oxygen of cell damage caused by the state.(3)Mitochondrial function detection:Two groups of cells based on cultivating 24 h and fixed slice,using transmission electron microscope under different oxygen time change.Intracellular reactive oxygen species(ROS)of detection the active oxygen detected by flow cytometry were used to detect the change of intracellular reactive oxygen content under each point in time.Mitochondrial respiratory chain core protein mitochondrial pigment C as the target protein,by Western blot detection of the expression of each time period,to determine the degree of damage to mitochondria.(4)Observation of apoptosis:The activity of caspase 3 was detected by ELISA.The cells were stained with fluorescent dye Hoest 33342 for 0h,24 h and 48 h,and the apoptotic cells were determined by fluorescence microscope.7-AAD and Annexin V-APC.The early apoptotic rate of the two groups was detected by flow cytometry.(8)Cell necrosis: The degree of expression of lactate dehydrogenase(LDH)at each time was measured by ELISA,and the extent of cell necrosis was determined as an index.Result(1)Cell growth and viability of observation:The growth rate of cardiomyocytes was slowed down with the increase of hypoxia time by cell counting.Compared with the control group,the cell growth rate of the two groups was the largest at 24 h,and the cell growth rate in the 24 h group was 1.53 times lower than that in the control group(P <0.01).The survival rate of cardiomyocytes was significantly decreased with the increase of hypoxia time using CCK-8 kit.The cell growth rate of the 6h group was 1.53 times lower than that of the control group(P <0.01).After 6 hours of hypoxia,the morphology of cardiomyocytes began to change.After 12 hours of hypoxia,the cytoplasm of cardiomyocytes appeared vacuoles and cell shrinkage.All cells died in hypoxia for 48 hours.(2)Mitochondrial function detection:24 hours of hypoxia showed significant damage to the structure of myocardial cell organelles.Hypoxia 6 hours,ROS release levels began to rise,24 h two groups of different intracellular ROS level,the largest oxygen 24 h group of intracellular ROS expression level increased 7.59 times(P < 0.01).Oxygen began in 3 hours difference,compared with normal group,hypoxic 3 h group of cytochrome C protein expression level increased 0.79 times(P< 0.01).Compared with 12 h group of oxygen,oxygen 24 h group of cytochrome C protein expression level increased 0.41 times(P < 0.01).(3)Observation of cell apoptosis: with the increase of oxygen time,caspase 3 activity level is also rising,oxygen began in 3 hours difference,compared with normal group,hypoxic group 3 h caspase 3 activity levels increased 0.67 times(P< 0.01).48 hours of oxygen activity,compared with 24 h group of oxygen,oxygen 48 h group of caspase 3 activity levels increased 0.27 times(P< 0.01).Observed by fluorescence microscope,anoxic cultured rat myocardial cells of oxygen treatment after 24 h,48 h,present typical morphological changes of apoptosis cells.By flow cytometry reflects oxygen increased with time and early apoptosis rate of myocardial cells was obviously increased.Oxygen 24 h cell apoptosis rate is highest,24 h compared with normal group,hypoxic group level of apoptosis increased 14.14 times(P< 0.01).(4)Cell necrosis:The release rate of lactate dehydrogenises(LDH)in cardiomyocytes was significantly increased with the increase of hypoxia time.The release rate of lactate dehydrogenises LDH in hypoxia group was higher than that in normal group The level increased by 3.1 times(P <0.01).Conclusion Hypoxia can cause the cell growth and survival rate of rat cardiac cell line(H9C2),the cell morphology is obviously changed and mitochondrial damage,mitochondrial damage can lead to apoptosis,hypoxia time can cause cell necrosis.