| Aflatoxin is a metabolite of aflatoxin and Aspergillus parasiticus, which is recognized as environmental pollutants. Aflatoxin pollution is a global public health problem that can be harmful to human health. In the area of tropical and subtropical, this situation is particularly serious. The aflatoxin B1 is the most common forms of AFT, the toxicity of AFB1 is the most strong, carcinogenic strongest. AFB1 can induce liver damage and liver cancer easily and it’s also considered as the Class I carcinogen. The aflatoxins in nature, the toxicity of AFB1 is the most strong, and the liver is the important target organs of poisoning, excessive intake by human body, AFB1 can damage the liver cell, cause acute hepatitis. Plant Polyphenols chemicals gradually to the attention of the people. Epigallocatechin Gallate (EGCG) as a major active constituent of green tea, antioxidant, anti mutation, induce cancer cells apoptosis, improving immunity, the effect of catechin compounds in biological effect is the strongest one.Procyanidin B2 (PC-B2) mainly comes from the plants, flowers, fruits and seeds of the plant kingdom class of widely exists in natural polyphenol compounds, heating in acid medium, the oxidation and degradation of anthocyanins of red, therefore is called the procyanidins. PC-B2 belongs to the strong reducing agent, it can remove people and animals of free radicals in the body, protect the cells and tissues from strong oxidizing free radical damage. Its pharmacological effects is extensive, protect cardiovascular, prevention of high blood pressure, antitumor, resistance to radiation, and so on. This study proposed by in vitro cell culture experiment platform, evaluation of EGCG with PC-B2 for the handling of the later generations to normal liver cell DNA damage by AFB1 protection.Part 1: The influence of AFB1 on the in vitro cultivation of L-02 cells survival rate, apoptosis rate and DNA damageObjective:To explore the different concentrations of AFB1 on the in vitro human embryonic liver cells (L-02) cell survival rate, apoptosis rate and the effect of DNA damage. Methods:in vitro cultivation of L-02 cells, AFB1 intervention, AFB1 drug concentrations are:0, 5, 10, 20, 40, 80mg/L AFB1. Inverted microscope to observe each cell morphological changes, determined by MTT method is used to test the cell survival rate, using flow cytometry instrument detecting apoptosis rate, single cell gel electrophoresis test (SCGE) are discussed to observe the cellular DNA damage. Results:compared with control group,10 mg/L or more concentration of AFB1 intervention after 48h cell morphological changes, low concentrations of cell shrinkage, smaller volume, high concentration of cells shrivel into round, appear a large number of cells and cell debris floating, the degree of morphological changes associated with AFB1 concentration.More than 10mg/L and concentration of AFB1 role after 48h survival rate were obviously lower than the control group (P<0.05); More than 5mg/L and AFB1 concentration effect after 48h apoptosis rate were significantly higher than that of control group (P<0.05); Concentration and 10 mg/L or more AFB1 intervention dose-effect relationship, with the increase of concentration of AFB1 role, L-02 cell survival rate reduced (P<0.05) and apoptosis rate increased (P<0.05).SCGE experiment results show that: compared with control group, AFB1 L-02 cells after 48h, the concentration of 10mg/L or more groups of cells increased percentage, Olive tail from comet tail DNA, the difference was statistically significant (P<0.05), and the dose-effect relationship, with the increase of AFB1 concentration, L-02 cell DNA damage increased.Conclusion: 10mg/L and above in AFB1 concentration cell toxicity, and inhibit the growth of cells, 5mg/L and above AFB1 concentration could increase the rate of apoptosis; With the increase of AFB1 concentration, the first dose-effect relationship.40mg/L AFB1 concentration can inhibit the growth of cells, and caused serious damage of the cellular DNA effectively, but not immediately kill cells, and 40mg/L concentration was selected as the reference concentration of follow-up test.Part 2:EGCG with PC-B2 to AFB1 cause L-02 cell survival rate, apoptosis rate and the effect of DNA damageObjective:To investigate the correlation between the EGCG and PC-B2 AFB1 to cultivate human embryonic liver cells in vitro (L-02), DNA damage and survival rate, apoptosis rate, the influence of the gene expression of Bcl-2, Bax, caspase-3, caspase-9 and P53 gene expression. Methods: in vitro culture of L-02 cell, the experiment set up six groups:(1) the blank control group:L-02 cells only give culture;(2) the solvent control group to give 2% DMSO solvent culture; (3) the AFB1 infected group: L-02 cells by 40mg/L AFB1 alone;(4) the EGCG groups:L-02 cells in AFB1 infected while giving 50mg/L EGCG effect;(5) the PC-B2 groups:L-02 cells at the same time in the AFB1 infected given 200mg/L PC-B2; (6) EGCG and PC-B2 groups:L-02 in AFB1 infected cells while giving 50mg/L EGCG and 200mg/L PC-B2. Above each liquid for a 2 days,3 to 5 days to extend once, after a week, inverted microscope to observe each cell morphological changes, determined by MTT method is used to test the cell survival rate, using flow cytometry instrument detecting apoptosis rate, single cell gel electrophoresis test (SCGE) are discussed to observe the cellular DNA damage, using Western blot method to analysis the Bcl-2, Bax, caspase 3, 5 caspase-9, P53 gene expression. Results:(1) compared with control group, AFB1 infected group could significantly inhibit the growth of liver cells, EGCG with PC-B2 can reduce L-02 cell growth inhibition caused by AFB1, the liver cell growth inhibition rate reduced from 61.12% to 42.18% and 46.72%; EGCG with PC-B2 combination makes AFB1 on liver cell growth inhibition rate from 61.12% to 37.15%. SCGE experimental results have shown that EGCG and PC-B2 combination can reduce DNA damage of L-02 cell caused by AFB1 (P<0.05), the comet Olive tail moment value of AFB1 infected group 9.72±2.64, EGCG group 5.91±1.79,PC-B2 group 5.36±1.17,EGCG+PC-B2 group 4.47±1.27 respectively. After treatment, the early apoptosis rate (%) of L-02 are as followings:blank control group 2.63±0.28, solvent control group 3.01±0.45, AFB1 infected group 13.63±3.79, EGCG group 8.26±1.67,PC-B2 group 7.89±1.52, EGCG+PC-B2 group 4.68±0.51 respectively, the experimental results show significant difference (P<0.05); Compared with solvent control group, the AFB1 infected group can raise caspase 3, the expression of caspase-9 (P<0.05), cut the BCL-2/Bax (P<0.05); And EGCG with PC-B2 group that EGCG with PC-B2 alone could cut caspase 3, the expression of caspase-9 (P<0.05), significantly raising the Bcl-2/Bax (P<0.05), while the EGCG in conjunction with PC-B2, the influence of caspase-3, caspase-9 and BCL-2/Bax are more significantly (P<0.05); Whether EGCG useing alone or combined with PC-B2, the level of expression of P53 was not obvious. Conclusion:AFB1 could significantly inhibit the growth of L-02 liver cells, and make the cell’s DNA damage degree aggravating;EGCG with PC-B2 alone or combined can improve the cell survival rate, reduce DNA damage and apoptosis rate and;EGCG with PC-B2 cells of AFB1 long time exposure of L-02 caspase 3, caspase-9 gene expression significantly lowered, a significant rise in the Bcl-2/Bax ratio, EGCG with PC-B2 can lower AFB1 L-02 cell DNA damage and apoptosis rate and its mechanism may be related to downgrade the expression of caspase-3, caspase-9, increases the Bcl-2/Bax ratio. |
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