| Melamine is a chemical raw material, because of its higher nitrogen, so it was incorporated into the milk or milk powder by lawless people in 2008. It hurt to the people, especially infants and young children and bring great damage. Melamine is nitrogen-containing heterocyclic compounds, it is likely to cause biological DNA damage. Tetrahymena is the class of protozoan cilia, is also a single-cell eukaryotes, its way of life in a free swim in fresh water, so the stress response to pollutants is more sensitive and more direct; further more its short life cycle ( 2 ~ 2.5 h) and easy to operate method of sterile culture, it has been used as a good model organism in toxicology studies. In order to understand the melamine toxicity to tetrahymena. The DNA damage was observed by using the microscope to observe; the melamine on the impact of cell reproduction and sexual reproduction with cell counting; with the comet assay and DAPI staining of nuclear technology used to observe the gene group changes was studied with single cell gel electrophoresis (SCGE); the cell apoptosis was analyised by flow cytometry; and then metallothionein gene expression changes was analyzed by fluorescence quantitative PCR. the specific findings are as follows:1. Melamine toxicity of Tetrahymena. The influence of melamine on the growth of Tetrahymena and mating was studied with concentration of 0, 1, 2, 3, 4 g / L melamine medium. Growth experiments showed that Tetrahymena growth was more depressed with the higher concentrations of melamine, the calculated IC50 was 1 g / L. Mating experiments results showed that compared with the control group, low concentration (1, 2 g / L) of melamine can promote Tetrahymena mating, 2the highest number of mating when the concentration is 2 g / L; later with the melamine concentration higher, the number of mating decreased, it indicate that the high concentration (3, 4 g / L) inhibited Tetrahymena mating. Show that the melamine could have damaging effects on cells.2. Tetrahymena genome influence and apoptosis by melamine. In this study, the DNA damage was studied by SCGE, the results indicate that with the higher concentration of melamine, the DNA damage of melamine on the Tetrahymena genome have also enhanced the extent, and found that the highest concentration of melamine (4 g / L) on Tetrahymena to produce significant damage to the genome. Treated cells apoptosis was anlaysised by flow cytometry and found that cells apoptosis appeared obvious at 2 g / L and 4 g / L concentration of melamine.3. Tetrahymena metallothionein MTT1 expression influence by melamine. Metallothionein gene has a protective effect for genome, in this study we analysised metallothionein gene expression change under the different melamine concentrations (0 g/ L, 1 g/ L, 2 g/ L, 4 g/ L) stress Tetrahymena last 1 h, the results showed: treatment group and control group cells, MTT1 gene was expressed; and MTT1 gene of Tetrahymena expressed in abundance with the increase of the concentration of melamine, the 4g/ L melamine treatment are the maximum, while in the 1 g / L melamine treatment, the expression level of the minimum. The cell apopotosis was analysise by the Flow cytometry, the results showed that low concentrations of melamine on the single-cell Eukaryotes less damage, but high concentrations of melamine in greater damage to the cells, it is idertical with the previous experimental results.To conclude, melamine can influence the single-cell Eukaryotes Tetrahymena propagation and sexual reproduction, and can result in Tetrahymena genome DNA damage at higher concentration of melamine.MTT1 gene expression intreased when Tetrahymena under melamine stress.
|
PDF Full Text Request