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DNA Damage of HUVECs Induced by Cholesterol and Protection Effect of Gypenosides

Author: WangHuiXing
Tutor: QianMinZhang
School: Zunyi Medical College,
Course: Biochemistry and Molecular Biology
Keywords: gypenosides DNA damage cholesterol γH2AX ROS
CLC: R285.5
Type: Master's thesis
Year: 2011
Downloads: 13
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Abstract


Objective:This work is devoted to investigate DNA damage of HUVECs induced by cholesterol and the protection effect of gypenosides.Methods:1.Human umbilical vein endothelial cell line CRL-1730 was cultured in vitro and identified with VIII fluorescent antibody.2.Three methods were used to examine yH2AX:after incubated with cholesterol at 12.5mg/L,25mg/L,50mg/L for 12 hours, the yH2AX foci formation in CRL-1730 cells was detected by immunofluorescence; the expressions of yH2AX protein was examined by western blot and flow cytometry(FCM). 3.After a one hour pre-treating with NAC or different dosages of gypenosides,CRL-1730 cells were incubated with cholesterol at 50mg/L for 12 hours. The yH2AX foci formation was detected by immunofluorescence and the yH2AX protein expressions was detected by western blot;the expressions of ROS were detected by FCM.Results:1. VIII factor immunofluorescence test shows positive endothelial cell dentity of the cultured CRL-1730 cells.2. After 12 hours cholesterol incubation,the yH2AX foci formation was able to be detected in all 3 cholesterol dosage group,which increases with the cholesterol dosage ascending.Western blot and FCM shows that the expression level of yH2AX protein increases along with the increase of cholesterol dosage,which are all significantly higher than that of the control group.3.In the NAC or gypenosides pre-treating group,the yH2AX foci formation decreases along with the increase of gypenosides dosage.Meantime,the yH2AX protein expression level decreases with the increase of gypenosides dosage.Both the yH2AX foci formation and the protein expression decrease significantly than that of the control group.FCM shows that ROS level decreases along with the gypenosides dosage increases,which is significantly lower than the control group.Conclusion:1.Cholesterol induces DNA damage in HUVECs by increasing ROS levels. 2.Gypenosides decreased DNA damage effect of cholesterol by lowering ROS levels.

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