| Background and ObjectiveAge related macμlar degeneration is a progressive eye disease which leading the course of vision loss or no vision in the center of the visual field.An increasing number of surveys showed that people will suffer retinal photodamage when they exposure to the high density of blue light in a long time.For example,Blue light can induced oxidative stress and apoptosis in retinal pigment epithelium(RPE)cell and mitochondria played a crucial role in RPE cell damage induced by blue light.But it is not clear what is the mitochondria and mitochondrial DNA-participated mechanism and changes in human RPE cell damage.To study the mitochondrial-participated mechanism of RPE cell damage and cell apoptosis induced by blue light,we separate human primary RPE cell and observe the cell damage with different duration of blue light exposure in vitro.MethodsHuman primary RPE cells were separated from fresh detachment eye ball,4th and 5th generation will be used in this examination with identification.Reformed the normal cell cμlture incubator with 5%CO2 atmosphere into a blue light incubator box,adjusted the blue light indensity by FL-1D blue light irradiatometer and made sure the idensity of blue light on RPE cell surface range at 4.0±0.5mW/cm2.The RPE cells were divided into 8 groups,including 0-hour light exposure group(control group),1/2-hour,1-hour,2-hour,4-hour,6-hour,12-hour and 24-hour light exposure group.According to different experiment requirements,cells were classified into different groups.Immunofluorescence was used to identified the cell;the expressions of Caspase-3、Cleaved Caspase-3、Caspase-9、Cleaved Caspase-9、Bax、Bcl-2 proteins was detected with western blot;RT-PCR was used to detect the expressions of Caspas-3、Caspase-9、Bax and Bcl-2 genes;qPCR was used to detect the copy number of Mitochondrial DNA and PCR was used for detedt Mitochondrial DNA 4977bp common deletion.PCR expansion will be sent to sequencing.Resμlts1.Western Blot showed the expressions of Bax were up-regμlating after illumination for 1h,Cleaved Caspase-3 were up-regμlating after illumination for 2h,Caspase-3、Caspase-9、Cleaved Caspase-9 were up-regμlating after illumination for 4h,while down-regμlating the expression of Bcl-2 after illuminated for 2h,the expressions was make significant differences compared to control group(P value<0.05).2.The RT-PCR showed that Bcl-2 were up-regμlating after illumination for 2h and decreased compared with blank group after illumination for 6h(P<0.05),Caspase-3、Caspase-9 and Bax were up-regμlating after illumination for 12h、4h and 4h respectively,the results showed statistically significant compared to control group(P value<0.05).3.The copy number of Mitochondrial DNA was down-regμlating compare to control group in a time-dependent fashion(P value<0.05).4.The expression of 4977bp common deletion were increased and the situation got worse with the extension of the blue light illumination time.5.Gene sequencing showed the PCR expansion produces match with the order of mitochrondial DNA after 4977bp common deletion.Conclusion1.The proteins expression of Bax increased after 1h blue light irradiation.The expression of Cleaved Caspase-3 began to increased accompanied the expression of Bcl-2 decreased.The expression of Caspase-3、Caspase-9.Cleaved Caspase-9 increased after 4h blue light irradiation,and the RT-PCR showed the same trend,which indicated apopotosis system was activated,and the mitochondria mediated apopotosis system played an very-important role in it.2.The copy number of Mitochondrial DNA was downregμlating with 2h blue light irradiation,and after 4h blue light exposure,mitochondrial DNA 4977bp deletion increased,which indicated blue light can influence of mitochondrial DNA replication and increase the mutation rate.Which induced mitochondrial DNA dysfunction when illumination time reached 2h,including replication decreased and the mutation rate increased;subsequently,it occur RPE cells damage,as well as cell apoptosis and cell death. |
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