Hypoxic Microenvironment Influence On Glioma Cells Mitochondrial Function

Part 1. Hypoxic microenvironment on glioma cell mitochondria permeability transition pore functionObjective:Mitochondrial membrane permeability into hole as mitochondrial membrane and outer membrane protein channel of a variety of protein, the mitochondria inside and outside material permeability plays an important role. Open MPTP can lead to a lot of smaller molecules into the mitochondrial membrane, mitochondria swelling, outer membrane rupture, is located in the mitochondrial cytochrome C between inner and outer membrane, such as cell apoptosis factors into the cytoplasm, cause apoptosis. This part studies by measuring the concentration of oxygen MPTP open situation, understand the effects of different oxygen concentration on the mitochondrial function.Methods:T98g glioma cells under constant oxygen training to traits stable expression, Three gas low oxygen incubator points,5%,0.1%, two different oxygen concentration, cultured cells. To extend the homologous generation cells dish in a petri dish, June 3 plate of low oxygen and oxygen often group of cells that correspond to the 24 h,48 h,72 h. In appropriate time to use the corresponding cells mitochondrial permeability transition pore kit after processing, the cells in the membrane permeability into hole under open conditions (excitation wavelength 488 nm).Results:T98g glioma cells under the condition of 5% of the low oxygen membrane permeability transition pore opening degree and often oxygen groups; 0.1% low oxygen groups more often oxygen MPTP openness significantly increased; Under the same low oxygen levels, cell hypoxia, the longer the higher the degree of membrane permeability transition pore opening. Often oxygen group, hypoxia group,5% and 0.1% hypoxia group 3 group of cells, under the same cultivation time, the lower the oxygen concentration, the membrane permeability transition pore opening degree is higher.Conclusion:T98g glioma cell lines under the moderate hypoxia microenvironment, a slight increase in mitochondrial permeability transition pore opening degree, low oxygen levels after further increased to extremely low oxygen, obviously increase the degree of the mitochondrial permeability transition pore opening.Part 2.Hypoxic microenvironment on glioma cell mitochondria permeability transition pore functionObjective:Mitochondrial membrane potential refers to and mitochondrial membrane potential difference on either side of the outer membrane and mitochondrial biofilm on both sides of the different ion concentration, reaction is more sensitive indicator of mitochondrial function integrity, this part of the research through the study of the mitochondrial membrane potential under different oxygen concentration.Understand the effects of different oxygen concentration on the mitochondrial function, from the cellular level for hypoxic microenvironment influence on T98g glioma cell apoptosis.Methods:T98g glioma cells expressed in often train to cellular stability under oxygen, low oxygen incubator points,5%,0.1%, two different oxygen concentration cultured cells. To extend the homologous with the generation of cells in different 12 orifice, low oxygen and oxygen often group 2 disc cells, each corresponding to the48 h,72 h, and useing 6cm cell culture cell culture dish, extractiing mitochondria from cell at 48 hours and 72 hours. At corresponding time after joining dilution to the cells of the corresponding JC-1 stain, incubation after 20 minutes, the laser confocal microscope (n= 485nm wavelength and n= 540 nm), and calculate the fluorescence intensity.Results:Under normal oxygen concentration, mitochondrial membrane potential T98g glioma cells, normal dyeing by JC-1 red; Hypoxia group was 5% more red laser microscope, a small amount of mitochondria green,0.1% low oxygen group of mitochondria after dyeing JC-1, mitochondrial green more, a small amount of mitochondria was red. Glioma cells under the condition of same low oxygen cultivation, the longer the mitochondria after dyeing JC-1 degree of green fluorescence.Conclusion:T98g glioma cell line in oxygen and 5% under hypoxic microenvironment, mitochondrial membrane potential is much red fluorescence, mitochondrial function is normal, and mitochondrial membrane potential under low oxygen 0.1% green, mitochondrial membrane potential decreased obviously, mitochondrial function declined obviously. T98g glioma cell line in oxygen and 5% under hypoxic microenvironment, mitochondrial membrane potential is much red fluorescence, mitochondrial function is normal, and mitochondrial membrane potential under low oxygen 0.1% green, mitochondrial membrane potential decreased obviously, mitochondrial function declined obviously.