杂色曲霉素引起Hep G2细胞DNA链断裂的氧化应激以及溶酶体膜通透机制的研究

发布时间：2019-03-05 16:24

【摘要】：目的：研究ST诱导的DNA损伤与细胞内溶酶体膜稳定性以及ROS水平的关系，旨在探讨ST遗传毒性的机制。方法：实验体系选定Hep G2细胞，为了评价ST的遗传毒性，首先通过利用单细胞凝胶电泳（SCGE）实验(彗星实验）来检测HepG2细胞DNA的损伤状况。为了进一步研究ST遗传毒性发生的途径，我们检测了细胞中ROS的水平，通过2’，7’ 二氢二氯荧光素（DCFH）法来完成，用荧光色素吖啶橙（Acridine orange，AO）测定细胞内溶酶体膜的稳定性，，用免疫组化技术分析ST诱导DNA中产生的8羟基脱氧鸟苷（8-OHdG）的水平；同时，分别采用氯化铵（NH4CL）和N-乙酰半胱氨酸（N-Acetylcysteine,NAC）干预ST所导致的DNA损伤。应用SPSSv11.5统计软件包对实验结果进行统计分析。结果：HepG2细胞在经过0.5~2μg/ml的ST染毒处理1h后，细胞溶酶体膜稳定性发生改变，通透性增加，细胞内ROS水平增加，8-OHdG表达水平增加，DNA链发生断裂，细胞形成拖尾，呈彗星样，且其尾长同对照组相比增长明显并且呈剂量依赖关系。与此同时，分别用10mM的NH4CL和NAC对HepG2细胞进行1h的预处理后，ST所引起的DNA损伤链断裂几乎完全被抑制。溶酶体内部的酸性环境维持了溶酶体膜的稳定性，经10mM的NH4CL预处理降低了细胞内的PH值后，ST所引起的Hep G2细胞溶酶体膜稳定性得到很好的保护；NAC是已知有效的抗氧化剂，在经过10mM的NAC预处理1h的干预试验后，明显降低了细胞内ROS产生的水平。结论：杂色曲霉素会导致Hep G2细胞DNA链发生断裂，对DNA造成损伤，具有DNA损伤毒性。其作用机制可能与溶酶体途径以及氧化应激途径有关。在ST的毒性作用下，溶酶体膜稳定性遭到破坏，从而释放一些酸性水解酶，导致DNA链断裂；ST也可能通过引起HepG2细胞发生氧化应激，导致细胞内ROS水平升高，DNA氧化损伤标志物8-OHdG表达增强，从而对DNA造成氧化性损伤。此外，NAC作为一种有效的抗氧化剂，减轻了ST对HepG2细胞所致的DNA链断裂，说明ST所引起的DNA损伤也可能是氧化性的损伤。
[Abstract]:Aim: to study the relationship between DNA damage induced by ST and lysosome membrane stability and ROS level in order to explore the mechanism of ST genotoxicity. Methods: in order to evaluate the genotoxicity of HepG2 cells, single cell gel electrophoresis (SCGE) assay (comet assay) was used to detect the damage of DNA in HepG2 cells. In order to further study the way of genotoxicity of ST, we detected the level of ROS in the cells, which was performed by the (DCFH) method of 2, 7', dichlorofluorescein dichloride, and the fluorescent pigment acridine orange (Acridine orange, was used. AO) was used to determine the stability of lysosome membrane and the level of 8-hydroxy-deoxyguanosine (8-OHdG) in DNA induced by ST was analyzed by immunohistochemistry. At the same time, ammonium chloride (NH4CL) and N-acetylcysteine (NAC) were used to interfere with DNA damage induced by ST, respectively. The experimental results were analyzed by SPSSv11.5 software package. Results: after HepG2 cells were treated with 0.5 ~ 2 渭 g / ml ST for 1 h, the membrane stability of lysosome changed, permeability increased, intracellular ROS level increased, 8-OHdG expression increased, DNA strand breaks, and cells formed tail-dragging. The tail length was significantly increased in a dose-dependent manner compared with the control group. At the same time, after pretreatment of HepG2 cells with NH4CL and NAC of 10mM for 1 h, the damage chain breaks of DNA induced by ST were almost completely inhibited. The lysosome inner acidic environment maintained the stability of lysosome membrane. After pretreatment with 10mM's NH4CL, the intracellular PH value was decreased, the lysosome membrane stability induced by ST was well protected from lysosome membrane in Hep G2 cells. NAC is known to be an effective antioxidant. After 1 h of pretreatment with NAC of 10mM, the level of intracellular ROS production was significantly decreased. Conclusion: the DNA strand breaks of Hep G2 cells can be induced by heterochromic aspergillus sp., which can damage DNA and have the toxicity of DNA damage. The mechanism may be related to lysosome pathway and oxidative stress pathway. Under the toxicity of ST, the stability of lysosome membrane was destroyed, and some acid hydrolases were released, which led to DNA strand break. ST may also cause oxidative damage to HepG2 cells by inducing oxidative stress in HepG2 cells, resulting in an increase in intracellular ROS level and an increase in the expression of 8-OHdG, an oxidative damage marker of DNA, thus causing oxidative damage to DNA. In addition, as an effective antioxidant, NAC alleviates ST-induced DNA strand breaks in HepG2 cells, suggesting that ST-induced DNA damage may also be oxidative damage.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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