Na-PCP诱导L-02肝细胞氧化应激和线粒体损伤的实验研究

发布时间：2018-01-13 05:32

本文关键词：Na-PCP诱导L-02肝细胞氧化应激和线粒体损伤的实验研究　出处：《中南大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的: 在体外试验系统(in vitro test),研究有机氯农药-五氯酚钠(Pentachlorophenol Sodium,Na-PCP)对L-02肝细胞氧化应激和线粒体损伤,为进一步了解五氯酚钠的毒作用机制奠定实验基础。方法: 以L-02肝细胞为受试细胞,通过MTT试验检测不同浓度Na-PCP对L-02肝细胞存活率的影响,选择细胞存活率为20%～90%的浓度进行后续实验。设置1个对照组和5个Na-PCP处理组,即12.812mg/L、19.218mg/L、25.624mg/L、32.030mg/L和38.436mg/L,处理时间为24h,采用化学比色法检测Na-PCP对L-02肝细胞超氧化物岐化酶(superoxide dismutase,SOD)活力、谷胱甘肽(reduced glutathione,GSH)和丙二醛(malondialdehyde,MDA)含量的影响;采用荧光分光光度法检测Na-PCP对L-02肝细胞线粒体膜电位(△Ψm)和线粒体通透性转运孔(permeability transition pore,PTP)的影响。设置1个对照组和9.609mg/L、12.812mg/L、19.218mg/L三个处理组,处理时间为24h,采用Western Blotting法检测Na-PCP对肝细胞p53、细胞色素C和凋亡诱导因子(apoptosis-inducing factor,AIF)的蛋白水平的影响。结果: 1.在9.609mg/L～38.436mg/L浓度范围内,Na-PCP能明显引起L-02肝细胞存活率的降低,且存在着浓度.反应关系(r=-0.978,P＜0.01)。 2.与对照组相比,12.812mg/L～38.436mg/L Na-PCP均可导致L-02肝细胞内MDA含量增加(r=0.956,P＜0.01),SOD活性下降(r=-0.900,P＜0.01),GSH含量减少(r=-0.975,P＜0.01)。 3.与对照组比较,在12.812mg/L～38.436mg/L浓度范围内,随着Na-PCP染毒浓度的增加,线粒体PTP开放度明显增大(P＜0.01),且PTP开放度与浓度之间呈现明显的浓度-反应关系(r=0.997,P＜0.01)。 4.与对照组比较,在12.812mg/L～38.436mg/L浓度范围内,Na-PCP导致荧光吸光度值升高,线粒体膜电位明显降低(P＜0.01),且膜电位的下降呈浓度依赖性(r=0.795,P＜0.01)。 5.经9.609mg/L、12.812mg/L和19.218mg/L Na-PCP处理L-02肝细胞,各组细胞p53、细胞色素C和AIF的蛋白水平明显高于对照组细胞(P＜0.05)。结论: Na-PCP在9.609mg/L～38.436mg/L的浓度范围内,能引起L-02肝细胞存活率下降,诱发氧化损伤,表现为SOD酶活力和GSH含量下降,MDA含量上升。Na-PCP导致肝细胞线粒体膜电位崩溃和通透性转运孔开放度增大,并使得细胞色素C和AIF从线粒体释放增多。Na-PCP导致p53蛋白水平增高。
[Abstract]:Objective:
In vitro test system (in vitro test), organochlorine pesticides (Pentachlorophenol - Sodium, Na-PCP of sodium pentachlorophenate) on L-02 hepatocyte oxidative stress and mitochondrial damage, provide an experimental basis for understanding the toxicity mechanism of pentachlorophenol.
Method:
In L-02 liver cells as test cells, the influence of the survival rate of L-02 cells was detected by MTT with different concentration of Na-PCP, the cell survival rate was 20% ~ 90% concentration for subsequent experiments. 1 control group and 5 Na-PCP group, 12.812mg/L, 19.218mg/L, 25.624mg/L, 32.030mg/L and 38.436mg/L, processing time 24h, detection of Na-PCP in L-02 cell superoxide dismutase by chemical colorimetric method (superoxide, dismutase, SOD) activity, glutathione (reduced glutathione GSH) and malondialdehyde (malondialdehyde, MDA) content influence; detection of Na-PCP by fluorescence spectrophotometry of L-02 hepatocyte mitochondria membrane potential (lpli m) and mitochondrial permeability transition pore (permeability transition, pore, PTP). The effects of 1 control group and 9.609mg/L, 12.812mg/L, 19.218mg/L three groups, treatment time is 24h, using Western Blotting method. The effect of Na-PCP on the protein level of p53, cytochrome C and apoptosis inducible factor (apoptosis-inducing factor, AIF) in liver cells.
Result:
1. in the range of 9.609mg/L ~ 38.436mg/L concentration, Na-PCP could significantly reduce the survival rate of L-02 hepatocytes, and there was a concentration. The reaction (r=-0.978, P < 0.01).
2., compared with the control group, 12.812mg/L to 38.436mg/L Na-PCP could increase the MDA content in L-02 cells (r=0.956, P < 0.01), SOD activity decreased (r=-0.900, P < 0.01), and the content of GSH decreased (r=-0.900, < 0.01).
3. compared with the control group, in the concentration range of 12.812mg/L to 38.436mg/L, with the increase of Na-PCP concentration, the mitochondrial PTP openness increased significantly (P < 0.01), and there was an obvious concentration response relationship between PTP openness and concentration (r=0.997, P < 0.01).
4. compared with the control group, in the concentration range of 12.812mg/L to 38.436mg/L, Na-PCP led to an increase in fluorescence absorbance and a decrease in mitochondrial membrane potential (P < 0.01), and the decrease of membrane potential was in a concentration dependent manner (r=0.795, P < 0.01).
5., 9.609mg/L, 12.812mg/L and 19.218mg/L Na-PCP were used to treat L-02 hepatocytes. The protein levels of p53, cytochrome C and AIF in each group were significantly higher than those in control group (P < 0.05).
Conclusion:
The concentration of Na-PCP in the range of 9.609mg/L to 38.436mg/L, L-02 can cause liver cell survival rate decreased, the oxidative damage induced by decreased SOD activity and GSH content, MDA content increased.Na-PCP induced hepatic mitochondrial membrane potential collapse and permeability transition pore opening degree increases, and the cytochrome C release from mitochondria increased.Na-PCP and AIF lead to increased p53 protein levels.

【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

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