Evaluation The Safety Of Exogenous Proteins By Cultured Cell Lines In Vitro

Background: With the increasing maturity of transgenic technology, it has been able to import the foreign genes into a variety of animal and plant objects to obtain the desired traits. The exogenous genes that insert into crops, is either a regulatory sequence, or encode a protein, many genetically modified crops that can realize people’s expectations are depend on the specific proteins. Prior to the introduction of the GM crops into the market, their safety needs to be thoroughly assessed. Currently, there is not a unified method that can be relied upon to predict the safety of exogenous proteins. Compared to the complicated models in vivo, the application of an invitro cell culture system for preliminary evaluating of GM foods has many advantages, such as low cost, time-efficient, less animal use, and obtain good results, to study cell mechanisms of induced cytotoxicity, finally the reproducibility of results. These advantages make it widely studied.Method: Choose ricin replace of foreign proteins, added to the cultured IM-9 cell lines, the concentration ranges of 0-100 ng / ml, in 6 h, 12 h check cells relative viability with CCK-8 to study the dose-time effect and make sure the concentration of half maximal inhibitory in 12 h. Add 20 ng / ml of ricin to induce cells. At 2 h, 6 h, 10 h, 12 h time points, detect the expression of genes TNF-α, CD40, IL-1β and Bcl-2, Bax, caspase. To make sure whether it is feasible to assess the safety by detection the genes expression of immune and apoptosis related; Use the comet assay, analysis test groups and control groups of genetic toxicity; AO / EB staining and Flow cytometry to detecte two groups of cell apoptosis. Draw a conclusion based on the experimental results.Results and Conclusion: The test groups, compared with the control in the 6 h time points, increase the relative viability of cells at lower doses(10~40 ng/m L), more than 40 ng/m L show inhibition effect. 12 h results showed that with the increase of the concentration, the relative viability of cells became lower, and the toxic effect was more obvious. Showed that the toxic affect was a certain dose-time effect. The results of gene expression detection showed that TNF-α, CD40 gene expressed significantly high in test groups. The percentage of tail DNA and OTM tail were compared by comet assay in the genetic toxicity test. They were significantly higher in the test groups and the apoptosis rates increased with the increasing of the concentration and time. Therefore, we can detect the relative cell proliferation rate, TNF- α and CD40 gene expression, comet assay, apoptosis detection to evaluation the safety of proteins by IM-9 cells.