黄曲霉毒素B_1致人胚肝细胞损伤的初步体外实验研究
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摘要
黄曲霉毒素(Aflatoxins,AF)是一类具有极强毒性的真菌次生代谢产物,对人类健康构成了非常严重的威胁。在天然污染的食品中以黄曲霉毒素B1(AflatoxinsB1,AFB1)最为多见,其毒性与致癌性最强。当人体大量摄入时,可引发急性中毒,出现急性肝炎、出血性肝坏死、肝细胞脂肪变性和胆管增生等。当微量持续摄入时,可造成慢性中毒,导致机体生长障碍、肝脏纤维性病变,引起肝癌等。针对以上特点,为进一步探索AFB1与肝损伤之间的关系,本课题进行了以下研究。
[目的]研究AFB1对人胚肝细胞(L-02细胞)的毒性作用特点,建立AFB1致L-02细胞DNA损伤的体外实验模型,为今后进一步探索对AFB1的预防性干预措施和机制提供参考依据。
[方法]以L-02细胞为靶细胞,采用完全随机设计的体外培养实验。对L-02细胞进行常规培养,选取处于对数生长期并生长良好的细胞进行试验。(1)MTT比色试验：试验设立空白对照及溶剂对照组、AFB1染毒组,分别用5、10、20、40、80、160、320mg/L七个剂量染毒。各组细胞培养24h后,进行MTT试验,观察各组细胞的活力及凋亡情况。(2)单细胞凝胶电泳试验(SCGE):试验设立空白及溶剂对照组、AFB1染毒组,分别用5、10、20、40mg/L四个剂量染毒。各组细胞培养24h后,收集细胞进行SCGE试验,观察细胞DNA损伤情况；(3)肝功能检测：同时收集细胞培养上清液,用全自动生化分析仪测定谷草转氨酶(AST)和谷丙转氨酶(ALT)含量。
[结果](1)MTT试验显示(?)0、160、320mg/L AFB1染毒组的吸光度A值与空白对照和溶剂对照组相比有显著性差异(P<0.01),细胞毒性指数(CI)达到或超过50%,呈现出一定的剂量-反应关系；(2)SCGE试验结果显示,浓度达到40mg/L AFB1的染毒组使细胞出现急性DNA损伤,其尾长(P<0.01)和Olive尾矩值(P<0.05)与空白对照和溶剂对照组相比有显著差异；(3)各AFB1染毒组的细胞培养上清液AST、ALT含量,分别与空白对照和溶剂对照组比较均未出现显著差异(P>0.05)。
[结论]MTT试验显示AFB1染毒浓度高于80mg/L时,方表现出显著的肝细胞毒性作用,使细胞存活率大大降低。SCGE试验中,AFB]染毒浓度达到40mg/L的浓度时,就能够诱导L-02细胞DNA出现较明显损伤,但同时肝功能AST和ALT却未见异常。表明低浓度AFB1染毒后,在未造成肝细胞急性损伤时,其致细胞DNA损伤的作用就已显现。提示我们在日常生活中如长期少量摄入AFB1,在未对机体造成明显的肝细胞凋亡和肝功能损伤之前,其致癌性就已表现出来。
由于以往国内外对AFB1的毒性作用研究多以动物实验为主或采用肝癌细胞进行体外实验,而使用人体正常肝细胞开展体外培养的实验研究甚少,因此在本次研究中我们选择L-02细胞为靶细胞,以AFB1为染毒物,探索AFB1致人胚肝细胞DNA损伤的相关剂量和敏感性指标,为今后进一步构建AFB1致肝细胞DNA损伤体外实验模型打下基础。
Aflatoxins is one kind has the greatly strengthened toxic Secondary Metabolites of fungus, has created extremely serious threat to the human health. In natural pollution food by Aflatoxin B1(AFB1) most sees, its toxicity and carcinogenicity is also strongest. When the human takes in massively, has the possibility to be able to have the acute poison, has the acute hepatitis, the hemorrhagic necrosis, the liver cell fat denaturation and the bile duct proliferation situation, etc. When micro continues to take in, may create the chronic poison, the growth barrier, causes the thready pathological change and the fiber structure proliferation, etc. In view of above characteristic, for further explores AFB1and between the liver damage relations, this topic has conducted following research.
Objective Discuss toxic effect to the Human embryo liver cell(L-02cell) induced by Aflatoxins B1, set up an in vitro model of L-02cell DNA injury, provide reference for study the preventive intervention of AFB1.
Methods Take the L-02cell as the target cell, uses the completely randomized design in vitro experiment. Carries on the routine culture to L-02cell, the selection is in the logarithmic growth phase and grows the good cell to carry on the experiment.(Ⅰ) MTT assay:The experiment sets up the blank control group, the solvent control group, the AFB1contamination group, with5,10,20,40,80,160,320mg/L seven dosage contaminations. Cultures after for24hours in each group of cells, carries on the cell MTT assay, observe the cell activity and apoptosis of each group.(II) Single cell gelelectrophoresis assay(SCGE):Grouping random experiment, sets up the blank and the solvent control group, the AFB1contamination group, with5,10,20,40mg/L four dosage contaminations. Cultures after for24hours in each group of cells, carries on SCGE assay, observe the situation of cell damage. At the same time, simultaneously collects the cell culture supernatant of each group, determines by the automatic biochemistry analyzer to analy aspartate aminotransferase (AST) and alanine aminotransferase (ALT).
Results (I) MTT assay showed that each exposure group compared with blank control significant difference (P<0.01), cell toxicity was dose-response relationship.(II)SCGE showed that only40mg/L exposure groups showing a cell damage, Taillength (P<0.01) and Olivetailmoment (P<0.05) compared with blank control were significantly different.(Ⅲ) The cell culture supernatant of each exposure group of AST and ALT there were no significant differences (P>0.05) compared with blank control.
Conclusion MTT assay of exposure doses above80mg/L or more, can present significant cytotoxic effect, cause the cell viability decreased significantly. SCGE have shown that40mg/L AFB1was able to induce the L-02cell DNA damage; but the value of AST and ALT had no abnormal finding. Test results indicate that DNA damage occurred when had not be found acute injury of cell induced by low concentration of AFB1. Intake less AFB1during prolonged would have induced cancer. AFB1induced acute injury of cultured cells in vitro at home and abroad are few reports of acute toxic effects have yet to be further explored, so chose L-02cell and AFB1in this research. The acute toxic effect of AFB1that needs to be further investigated research, in order to set up in vitro cell model.
[目的]研究AFB1对人胚肝细胞(L-02细胞)的毒性作用特点,建立AFB1致L-02细胞DNA损伤的体外实验模型,为今后进一步探索对AFB1的预防性干预措施和机制提供参考依据。
[方法]以L-02细胞为靶细胞,采用完全随机设计的体外培养实验。对L-02细胞进行常规培养,选取处于对数生长期并生长良好的细胞进行试验。(1)MTT比色试验：试验设立空白对照及溶剂对照组、AFB1染毒组,分别用5、10、20、40、80、160、320mg/L七个剂量染毒。各组细胞培养24h后,进行MTT试验,观察各组细胞的活力及凋亡情况。(2)单细胞凝胶电泳试验(SCGE):试验设立空白及溶剂对照组、AFB1染毒组,分别用5、10、20、40mg/L四个剂量染毒。各组细胞培养24h后,收集细胞进行SCGE试验,观察细胞DNA损伤情况；(3)肝功能检测：同时收集细胞培养上清液,用全自动生化分析仪测定谷草转氨酶(AST)和谷丙转氨酶(ALT)含量。
[结果](1)MTT试验显示(?)0、160、320mg/L AFB1染毒组的吸光度A值与空白对照和溶剂对照组相比有显著性差异(P<0.01),细胞毒性指数(CI)达到或超过50%,呈现出一定的剂量-反应关系；(2)SCGE试验结果显示,浓度达到40mg/L AFB1的染毒组使细胞出现急性DNA损伤,其尾长(P<0.01)和Olive尾矩值(P<0.05)与空白对照和溶剂对照组相比有显著差异；(3)各AFB1染毒组的细胞培养上清液AST、ALT含量,分别与空白对照和溶剂对照组比较均未出现显著差异(P>0.05)。
[结论]MTT试验显示AFB1染毒浓度高于80mg/L时,方表现出显著的肝细胞毒性作用,使细胞存活率大大降低。SCGE试验中,AFB]染毒浓度达到40mg/L的浓度时,就能够诱导L-02细胞DNA出现较明显损伤,但同时肝功能AST和ALT却未见异常。表明低浓度AFB1染毒后,在未造成肝细胞急性损伤时,其致细胞DNA损伤的作用就已显现。提示我们在日常生活中如长期少量摄入AFB1,在未对机体造成明显的肝细胞凋亡和肝功能损伤之前,其致癌性就已表现出来。
由于以往国内外对AFB1的毒性作用研究多以动物实验为主或采用肝癌细胞进行体外实验,而使用人体正常肝细胞开展体外培养的实验研究甚少,因此在本次研究中我们选择L-02细胞为靶细胞,以AFB1为染毒物,探索AFB1致人胚肝细胞DNA损伤的相关剂量和敏感性指标,为今后进一步构建AFB1致肝细胞DNA损伤体外实验模型打下基础。
Aflatoxins is one kind has the greatly strengthened toxic Secondary Metabolites of fungus, has created extremely serious threat to the human health. In natural pollution food by Aflatoxin B1(AFB1) most sees, its toxicity and carcinogenicity is also strongest. When the human takes in massively, has the possibility to be able to have the acute poison, has the acute hepatitis, the hemorrhagic necrosis, the liver cell fat denaturation and the bile duct proliferation situation, etc. When micro continues to take in, may create the chronic poison, the growth barrier, causes the thready pathological change and the fiber structure proliferation, etc. In view of above characteristic, for further explores AFB1and between the liver damage relations, this topic has conducted following research.
Objective Discuss toxic effect to the Human embryo liver cell(L-02cell) induced by Aflatoxins B1, set up an in vitro model of L-02cell DNA injury, provide reference for study the preventive intervention of AFB1.
Methods Take the L-02cell as the target cell, uses the completely randomized design in vitro experiment. Carries on the routine culture to L-02cell, the selection is in the logarithmic growth phase and grows the good cell to carry on the experiment.(Ⅰ) MTT assay:The experiment sets up the blank control group, the solvent control group, the AFB1contamination group, with5,10,20,40,80,160,320mg/L seven dosage contaminations. Cultures after for24hours in each group of cells, carries on the cell MTT assay, observe the cell activity and apoptosis of each group.(II) Single cell gelelectrophoresis assay(SCGE):Grouping random experiment, sets up the blank and the solvent control group, the AFB1contamination group, with5,10,20,40mg/L four dosage contaminations. Cultures after for24hours in each group of cells, carries on SCGE assay, observe the situation of cell damage. At the same time, simultaneously collects the cell culture supernatant of each group, determines by the automatic biochemistry analyzer to analy aspartate aminotransferase (AST) and alanine aminotransferase (ALT).
Results (I) MTT assay showed that each exposure group compared with blank control significant difference (P<0.01), cell toxicity was dose-response relationship.(II)SCGE showed that only40mg/L exposure groups showing a cell damage, Taillength (P<0.01) and Olivetailmoment (P<0.05) compared with blank control were significantly different.(Ⅲ) The cell culture supernatant of each exposure group of AST and ALT there were no significant differences (P>0.05) compared with blank control.
Conclusion MTT assay of exposure doses above80mg/L or more, can present significant cytotoxic effect, cause the cell viability decreased significantly. SCGE have shown that40mg/L AFB1was able to induce the L-02cell DNA damage; but the value of AST and ALT had no abnormal finding. Test results indicate that DNA damage occurred when had not be found acute injury of cell induced by low concentration of AFB1. Intake less AFB1during prolonged would have induced cancer. AFB1induced acute injury of cultured cells in vitro at home and abroad are few reports of acute toxic effects have yet to be further explored, so chose L-02cell and AFB1in this research. The acute toxic effect of AFB1that needs to be further investigated research, in order to set up in vitro cell model.
引文
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