农达（41％草甘膦）对人L-02肝细胞损伤的研究
发布时间：2018-11-18 15:21
【摘要】： 目的: 探讨农达(41%草甘膦)对人L-02肝细胞产生的损伤作用及其可能的机制。 方法: 体外试验(in vitro test)以L-02肝细胞为受试细胞,通过MTT法检测不同浓度的农达对L-02肝细胞存活率的影响,选择细胞存活率为20%-80%的浓度进行后续实验。设置5个农达处理组,60mg/L、90mg/L、120mg/L、150mg/L、180mg/L和1个阴性对照组(PBS),农达处理细胞时间为24h。L-02肝细胞的形态学改变采用姬母萨染色法在光镜下观察结合透射电镜观察;农达对L-02肝细胞膜通透性影响和细胞毒性作用采用谷草转氨酶(AST)、谷丙转氨酶(ALT)活性水平结合台盼蓝拒染法来评价;农达诱导L-02肝细胞氧化损伤程度选用超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽(GSH)含量等指标评价;用线粒体跨膜电位(△Ψm)检测细胞线粒体损伤;用DNA条带(DNA-Ladder)检测肝细胞DNA损伤;以AnnexinV-FITC/PI复染法测定细胞发生凋亡和坏死的情况;Western Blotting检测对照组与90mg/L组细胞色素C(Cyt C)和凋亡诱导因子(AIF)表达水平。 结果: 1.在60～180mg/L的处理浓度范围内,能明显引起L-02肝细胞存活率的降低(P＜0.05),处理浓度和细胞存活率之间存在负相关(r=-0.974)。 2.在光镜下观察姬母萨染色L-02肝细胞爬片,观察到各农达处理组L-02肝细胞皱缩或肿大,形态由瓦片状收缩为圆形,细胞间隙扩大,细胞破裂,染色质浓缩,贴壁细胞密度和数量减少。电镜下观察,可发现处理组细胞出现表面微绒毛消失,细胞膜结构不完整,细胞核碎裂或肿胀,线粒体空泡样变等细胞凋亡或坏死的表现。 3.农达处理L-02肝细胞,其活细胞台盼兰蓝染比例升高(P＜0.05);细胞培养上清液中ALT活性增加(P＜0.05);从90mg/L组开始AST活性增加(P＜0.05);农达导致处理组细胞内MDA的含量增加,SOD活性减弱,GSH含量减少(P＜0.05);农达导致120～180mg/L组细胞Na~+-K~+ ATP酶活性降低(P＜0.05)。 4.农达处理能诱导细胞DNA链断裂,处理浓度在150mg/L、180mg/L,DNA受损程度严重;90mg/L组Cyt C和AIF表达水平高于对照组(P＜0.05);90mg/L组开始用农达处理的L-02细胞线粒体膜电位明显降低(P＜0.05);农达能明显诱导处理组肝L-02细胞发生凋亡和坏死(P＜0.05),随着处理农度的增高,细胞凋亡和坏死的比例增高,但是在180mg/L组凋亡比例下降。 结论: 农达在60mg/L～180mg/L范围内,能引起L-02肝细胞存活率下降,细胞膜通透性增加,抑制细胞离子转运,诱发DNA损伤,线粒体膜电位降低,Cyt C、AIF等凋亡因子泄漏,使细胞产生凋亡和坏死,对肝细胞具有明显的损伤作用;其损伤的作用机制可能与农达导致肝细胞氧化损伤、线粒体崩溃等途径有关。
[Abstract]:Aim: to investigate the damage effect of Nongda (41% glyphosate) on human L-02 hepatocytes and its possible mechanism. Methods: L-02 hepatocytes were used as experimental cells in (in vitro test) in vitro. The effects of different concentrations of Nongda on the survival rate of L-02 hepatocytes were detected by MTT assay. The cell survival rate was 20%-80%. Set up five Nongda treatment groups, 60 mg / L, 90 mg / L, 120 mg / L, 150 mg / L, 180 mg / L, and one negative control group, (PBS), Morphological changes of hepatocytes treated with Nunda for 24h.L-02 were observed under light microscope and transmission electron microscope by Giemsa staining. The effects of Nongda on the permeability and cytotoxicity of L-02 liver cell membrane were evaluated by trypan blue exclusion method and the activity level of glutamic oxaloacetic transaminase (AST),) and alanine aminotransferase (ALT). The degree of oxidative damage of L-02 hepatocytes induced by Nongda was evaluated by superoxide dismutase (SOD),) malondialdehyde (MDA),) glutathione (GSH) content and mitochondrial transmembrane potential (蠄 m). DNA bands (DNA-Ladder) were used to detect DNA damage in hepatocytes and apoptosis and necrosis of hepatocytes were detected by AnnexinV-FITC/PI staining. The levels of cytochrome C (Cyt C) and apoptosis-inducing factor (AIF) were detected by Western Blotting in control group and 90mg/L group. Results: 1. The survival rate of L-02 hepatocytes was significantly decreased in the range of 60~180mg/L treatment (P < 0. 05), and there was a negative correlation between the treatment concentration and the cell survival rate (r-0. 974). 2. Under the light microscope, the L-02 hepatocytes were stained by Giemsa, and the L-02 hepatocytes in each Nanda treatment group were observed to shrink or swell, the shape of L-02 hepatocytes contracted from tile to round, the cell space was enlarged, the cells broke down, and the chromatin was concentrated. The density and number of adherent cells decreased. Under the electron microscope, it was found that the cells in the treated group showed apoptosis or necrosis, such as the disappearance of microvilli, incomplete cell membrane structure, fragmentation or swelling of the nucleus, vacuolar degeneration of mitochondria, and so on. 3. The percentage of trypan blue staining in L-02 hepatocytes was increased (P < 0. 05), the activity of ALT in supernatant of cell culture was increased (P < 0. 05), the activity of AST was increased from 90mg/L group (P < 0. 05). Nunda induced the increase of MDA content, the decrease of SOD activity and the decrease of GSH content (P < 0. 05), and the decrease of Na~-K-ATP enzyme activity in 120~180mg/L group (P < 0. 05). 4. Nunda treatment could induce DNA strand break, and the Cyt C and AIF expression levels in 90mg/L group were significantly higher than those in control group (P < 0. 05). The mitochondrial membrane potential of L-02 cells treated with nunda was significantly decreased in 90mg/L group (P < 0. 05). Apoptosis and necrosis of L-02 cells were induced by nongdanone significantly (P < 0. 05). With the increase of the degree of treatment, the proportion of apoptosis and necrosis increased, but the proportion of apoptosis decreased in 180mg/L group. Conclusion: nunda can decrease the survival rate of L-02 hepatocytes, increase cell membrane permeability, inhibit cell ion transport, induce DNA damage and decrease mitochondrial membrane potential (, Cyt C,) in the range of 60mg/L~180mg/L. The leakage of apoptosis factors such as AIF caused apoptosis and necrosis of hepatocytes, which had obvious damage effect on hepatocytes. The mechanism of the damage may be related to the oxidative damage of hepatocytes induced by Nunda and the breakdown of mitochondria.
【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
本文编号：2340410
[Abstract]:Aim: to investigate the damage effect of Nongda (41% glyphosate) on human L-02 hepatocytes and its possible mechanism. Methods: L-02 hepatocytes were used as experimental cells in (in vitro test) in vitro. The effects of different concentrations of Nongda on the survival rate of L-02 hepatocytes were detected by MTT assay. The cell survival rate was 20%-80%. Set up five Nongda treatment groups, 60 mg / L, 90 mg / L, 120 mg / L, 150 mg / L, 180 mg / L, and one negative control group, (PBS), Morphological changes of hepatocytes treated with Nunda for 24h.L-02 were observed under light microscope and transmission electron microscope by Giemsa staining. The effects of Nongda on the permeability and cytotoxicity of L-02 liver cell membrane were evaluated by trypan blue exclusion method and the activity level of glutamic oxaloacetic transaminase (AST),) and alanine aminotransferase (ALT). The degree of oxidative damage of L-02 hepatocytes induced by Nongda was evaluated by superoxide dismutase (SOD),) malondialdehyde (MDA),) glutathione (GSH) content and mitochondrial transmembrane potential (蠄 m). DNA bands (DNA-Ladder) were used to detect DNA damage in hepatocytes and apoptosis and necrosis of hepatocytes were detected by AnnexinV-FITC/PI staining. The levels of cytochrome C (Cyt C) and apoptosis-inducing factor (AIF) were detected by Western Blotting in control group and 90mg/L group. Results: 1. The survival rate of L-02 hepatocytes was significantly decreased in the range of 60~180mg/L treatment (P < 0. 05), and there was a negative correlation between the treatment concentration and the cell survival rate (r-0. 974). 2. Under the light microscope, the L-02 hepatocytes were stained by Giemsa, and the L-02 hepatocytes in each Nanda treatment group were observed to shrink or swell, the shape of L-02 hepatocytes contracted from tile to round, the cell space was enlarged, the cells broke down, and the chromatin was concentrated. The density and number of adherent cells decreased. Under the electron microscope, it was found that the cells in the treated group showed apoptosis or necrosis, such as the disappearance of microvilli, incomplete cell membrane structure, fragmentation or swelling of the nucleus, vacuolar degeneration of mitochondria, and so on. 3. The percentage of trypan blue staining in L-02 hepatocytes was increased (P < 0. 05), the activity of ALT in supernatant of cell culture was increased (P < 0. 05), the activity of AST was increased from 90mg/L group (P < 0. 05). Nunda induced the increase of MDA content, the decrease of SOD activity and the decrease of GSH content (P < 0. 05), and the decrease of Na~-K-ATP enzyme activity in 120~180mg/L group (P < 0. 05). 4. Nunda treatment could induce DNA strand break, and the Cyt C and AIF expression levels in 90mg/L group were significantly higher than those in control group (P < 0. 05). The mitochondrial membrane potential of L-02 cells treated with nunda was significantly decreased in 90mg/L group (P < 0. 05). Apoptosis and necrosis of L-02 cells were induced by nongdanone significantly (P < 0. 05). With the increase of the degree of treatment, the proportion of apoptosis and necrosis increased, but the proportion of apoptosis decreased in 180mg/L group. Conclusion: nunda can decrease the survival rate of L-02 hepatocytes, increase cell membrane permeability, inhibit cell ion transport, induce DNA damage and decrease mitochondrial membrane potential (, Cyt C,) in the range of 60mg/L~180mg/L. The leakage of apoptosis factors such as AIF caused apoptosis and necrosis of hepatocytes, which had obvious damage effect on hepatocytes. The mechanism of the damage may be related to the oxidative damage of hepatocytes induced by Nunda and the breakdown of mitochondria.
【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
【引证文献】
相关期刊论文 前1条
1 陈学梅;胡志军;陈建秋;;二氧化钛平推流反应器光催化矿化草甘膦研究[J];水处理技术;2013年03期
，本文编号：2340410
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2340410.html
最近更新
教材专著
