| Objective:The present study was designed to research and clarify the change condition of autophagy role in rats ovarian granulosa cells and PC12 cells in vitro after cadmium exposure. The other aim is to provide a scientific basis for further research about ovarian toxicity mechanism of cadmium.Methods:1.Research about autophagy in rat ovarian granulosa cells exposured to cadmium:The 21-day-old female SD rats' ovarian granulosa cells were extracted under the sterile condition in vitro,and they were divided into four groups randomly. When the cells were adherent stably and their shapes were stable,they were exposured to Cd(Cd Cl2) at different does of 0 ?M, 5 ?M, 10 ?M, 20 ?M in the time of 12 h. Then completed the following detections after the exposure ended:Transmission electron microscope was used to observate morphology changes and autophagosome formation in ovarian granular cells; Real time PCR method were used to detect the m RNA of MAP1LC3 b expression level in ovarian granulosa cells; Western-blot method were used to detecte LC3?/LC3 I protein expression levels in ovarian granular cells; Real time PCR method were used to detect the m RNA of Becn1 expression level in ovarian granulosa cells;Western-blot method were used to detecte Beclin-1 protein expression levels in ovarian granular cells.2.Research about autophagy in PC12 cells exposured to cadmium(Positive control verification experiment): PC12 cells were cultured and divided into two groups randomly, when the cells were adherent stably and their shapes were stable. Then they were exposured to Cd(Cd Cl2) at different does of 0 ?M and 20 ?M in the time of 4 h.Then completed the following detections after the exposure ended: Transmission electron microscope was used to observate morphology changes and autophagosome formation in ovarian granular cells; Western-blot method were used to detecte LC3?/LC3 I and Beclin-1 protein expression levels in ovarian granular cells.Results:1. Results about autophagy in rat ovarian granulosa cells exposured to cadmium:(1)Transmission electron microscope revealed that all group cells were not found autophagosome, while compared with control group the cells exposured to cadmium were found increase of apoptosis body. The phenomenon that ovarian granulosa cell shape is irregular and that chromatin abnormalities and that mitochondria swelled and cavitated increesed as cadmium dose increasing(10 ?M~20 ?M Cd).(2)The m RNA of LC3 expression level in rat ovarian granulosa cells exposured to Cd at different does of0 ?M, 5 ?M, 10 ?M, 20 ?M were 1.007, 1.027, 0.800 and 1.028, and the differences among all dose groups had no statistical significance(F=1.603, p>0.05); LC3? protein expression levels in rat ovarian granulosa cells exposured to Cd at different does of 0?M, 5 ?M, 10 ?M, 20 ?M were 0.603, 0.707, 0.787 and 0.577, and the differences among all dose groups had no statistical significance(F=0.480, p>0.05); LC3 I protein expression levels in rat ovarian granulosa cells exposured to Cd at different does of 0?M, 5 ?M, 10 ?M, 20 ?M were 0.433, 0.527, 0.560 and 0.437, and the differences among all dose groups had no statistical significance(?2=2.707, p>0.05); LC3?/LC3 I protein expression levels in rat ovarian granulosa cells exposured to Cd at different does of 0 ?M,5 ?M,10 ?M,20 ?M were 1.371,1.317,1.379 and 1.293,and the differences among all dose groups had no statistical significance(F=0.106, p>0.05).(3)The m RNA of Becn1 expression level in rat ovarian granulosa cells exposured to Cd at different does of 0 ?M, 5 ?M, 10 ?M, 20 ?M were 1.020, 1.160, 0.917, 0.870, and the differences among all dose groups had no statistical significance(F=1.170, p>0.05); and that Beclin-1 protein expression levels in rat ovarian granulosa cells exposured to Cd at different does of 0 ?M, 5 ?M, 10 ?M, 20 ?M were 0.570, 0.447, 0.577 and 0.410, and the differences among all dose groups had no statistical significance(F=0.676, p>0.05).2.Results about autophagy in PC12 cells exposured to cadmium(Positive control verification experiment): Under our experimental conditions,autophagy role of PC12 cells exposured to cadmium showed:(1)Transmission electron microscope revealed that compared with control group the cells exposured to cadmium were found increase ofisolated autophagosome with double membrane structure which contain organelles and cellular debris. Meanwhile the phenomenon that chromatin abnormalities and that mitochondria swelled and cavitated occured as cadmium exposure.(2)LC3? protein expression levels in PC12 cells exposured to Cd at different does of 0 ?M and 20 ?M were 0.655 and 0.970, and were significantly increased(t=8.707, p<0.01); LC3 I protein expression levels in PC12 cells exposured to Cd at different does of 0 ?M and 20 ?M were 0.350 and 0.827, and were significantly increased(t=6.154, p<0.01). LC3?/LC3? protein expression levels in PC12 cells exposured to Cd at different does of 0 ?M and 20 ?M were 0.535 and 0.851, and were significantly increased(t=7.190, p<0.01).Beclin-1 protein expression levels in PC12 cells exposured to Cd at different does of 0?M and 20 ?M were 0.535 and 0.851, and were significantly increased(t=3.227,p<0.05).Conclusions:1.Under the condition of this experiment, cadmium exposure can cause obvious damage of ovarian granular cells' shape and structure, but cell autophagy can not change. Meanwhile the pathway related to Beclin-1 does not change.2.Under the condition of this experiment, cadmium exposure can cause obvious damage of PC12 cells' structure, and can promote cell autophagy. Meanwhile the pathway related to Beclin-1 may play an important role of regulation in the change of autophagy induced by cadmium in PC12 cells. |
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