農達（41％草甘膦）對人L-02肝細胞損傷的研究
發(fā)布時間：2018-11-18 15:21
【摘要】： 目的: 探討農達(41%草甘膦)對人L-02肝細胞產生的損傷作用及其可能的機制。 方法: 體外試驗(in vitro test)以L-02肝細胞為受試細胞,通過MTT法檢測不同濃度的農達對L-02肝細胞存活率的影響,選擇細胞存活率為20%-80%的濃度進行后續(xù)實驗。設置5個農達處理組,60mg/L、90mg/L、120mg/L、150mg/L、180mg/L和1個陰性對照組(PBS),農達處理細胞時間為24h。L-02肝細胞的形態(tài)學改變采用姬母薩染色法在光鏡下觀察結合透射電鏡觀察;農達對L-02肝細胞膜通透性影響和細胞毒性作用采用谷草轉氨酶(AST)、谷丙轉氨酶(ALT)活性水平結合臺盼藍拒染法來評價;農達誘導L-02肝細胞氧化損傷程度選用超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽(GSH)含量等指標評價;用線粒體跨膜電位(△Ψm)檢測細胞線粒體損傷;用DNA條帶(DNA-Ladder)檢測肝細胞DNA損傷;以AnnexinV-FITC/PI復染法測定細胞發(fā)生凋亡和壞死的情況;Western Blotting檢測對照組與90mg/L組細胞色素C(Cyt C)和凋亡誘導因子(AIF)表達水平。 結果: 1.在60～180mg/L的處理濃度范圍內,能明顯引起L-02肝細胞存活率的降低(P＜0.05),處理濃度和細胞存活率之間存在負相關(r=-0.974)。 2.在光鏡下觀察姬母薩染色L-02肝細胞爬片,觀察到各農達處理組L-02肝細胞皺縮或腫大,形態(tài)由瓦片狀收縮為圓形,細胞間隙擴大,細胞破裂,染色質濃縮,貼壁細胞密度和數量減少。電鏡下觀察,可發(fā)現處理組細胞出現表面微絨毛消失,細胞膜結構不完整,細胞核碎裂或腫脹,線粒體空泡樣變等細胞凋亡或壞死的表現。 3.農達處理L-02肝細胞,其活細胞臺盼蘭藍染比例升高(P＜0.05);細胞培養(yǎng)上清液中ALT活性增加(P＜0.05);從90mg/L組開始AST活性增加(P＜0.05);農達導致處理組細胞內MDA的含量增加,SOD活性減弱,GSH含量減少(P＜0.05);農達導致120～180mg/L組細胞Na~+-K~+ ATP酶活性降低(P＜0.05)。 4.農達處理能誘導細胞DNA鏈斷裂,處理濃度在150mg/L、180mg/L,DNA受損程度嚴重;90mg/L組Cyt C和AIF表達水平高于對照組(P＜0.05);90mg/L組開始用農達處理的L-02細胞線粒體膜電位明顯降低(P＜0.05);農達能明顯誘導處理組肝L-02細胞發(fā)生凋亡和壞死(P＜0.05),隨著處理農度的增高,細胞凋亡和壞死的比例增高,但是在180mg/L組凋亡比例下降。 結論: 農達在60mg/L～180mg/L范圍內,能引起L-02肝細胞存活率下降,細胞膜通透性增加,抑制細胞離子轉運,誘發(fā)DNA損傷,線粒體膜電位降低,Cyt C、AIF等凋亡因子泄漏,使細胞產生凋亡和壞死,對肝細胞具有明顯的損傷作用;其損傷的作用機制可能與農達導致肝細胞氧化損傷、線粒體崩潰等途徑有關。
[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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R363
本文編號：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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R363
【引證文獻】
相關期刊論文 前1條
1 陳學梅;胡志軍;陳建秋;;二氧化鈦平推流反應器光催化礦化草甘膦研究[J];水處理技術;2013年03期
，本文編號：2340410
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