Induced Combination Apoptosis by FB1 and AFB1 in Vero Cell
|School||Nanjing Agricultural College|
|Course||Clinical Veterinary Medicine|
|Keywords||FB1 AFB1 apoptosis combined toxicity|
Fumonisin is a water-soluble mycotoxin which produced by Fusarium mycotoxins, which FBi is a major component, but also lead to the main toxicity of fumonisin. It was wildly contaminated in corn and their production, which has been to potential threaten human health and the development of animal husbandry. In vivo and in vitro studies, FB1’ toxic effect has been able to conform, including liver, kidney toxicity, pulmonary edema, neurotoxicity, immune suppression, and other carcinogenic. The results show that FB1 also has comparatively strong cytotoxicity, however now the report about FB1 on cytotoxicity studies is less, apoptosis induced by specific signal transduction mechanisms are not many reports. Meanwhile, AFB1 is a kind secondary metabolites which produced with Aspergillus flavus and parasitic aspergillus, and also present known toxicity strongest toxins, also exist widely in all sorts of grain and feed, but also cause mutation function and cause DNA damage effect, AFB1 also can cause induce sexual liver cancer, liver extensive hemorrhage, necrosis, on the kidneys are obvious damage effect. In view of two kinds of toxin exist widely in all sorts of grain and its products, and can produce kidney toxicity, so the experiment use FB1 sensitive cells monkey kidney cells Vero as the research object, was exposed with FB1 and AFB1 alone and the combined action to study its influence to morphology, cell activity, the mitochondrial membrane potential and apoptosis relevant protein expression, to discusses the impact of the changes that two kinds of toxins inducing apoptosis mechanism of action, and both of combined action, for further research on FB1 toxicity, and the mechanism FB1 induced apoptosis laid a foundation。TEST 1:In the present experiment, exponential phase of growth of Vero cells were exposed to different concentrations of FB1 and AFB1, and both of combined concentration, and setting a blank medium as control group. After 24h, using the MTT method for determination of a concentration group of cells Vero cell toxicity; the morphological changes of the Vero cells were observed under inverted with phase contrast microscope; DNA integrity were detected with agarose gel electrophoresis.The result shows:The MTT method for determination of toxins Vero cell proliferation inhibition rate, in same time, FB1 and AFB1 two toxin alone, with the toxin concentration effect increase, cell proliferation inhibition rate also gradually increase, When FB1 respectively with two concentration joint AFB1, FB1 cell proliferation inhibition rate than alone the inhibition rate are high, its IC50 also significantly reduced.Morphologic changes show that, from 2500 ng·mL-1 FB1 start to produce more apparent apoptosis effect to Vero cells, AFB1 15,30 ng·mL-1 two concentration on Vero cells have apoptosis-oriented role among them each concentration toxins group cell apoptosis rate are significantly higher than the control group(P< 0.05). Agarose gel electrophoresis, each group of cells are formed DNA ladder, proof of DNA cells have happened rupture, and caused a cell apoptosis.TEST 2:In the present experiment, Exponential phase of growth of Vero cells were exposed to different concentrations of FB1 and AFB1, and both of combined concentration, and setting a blank medium as control group. After 24h, then stained by JC-1, with flow cytometric analysis to detect the mitochondrial membrane potential changes.The result shows:The mitochondrial membrane potential detection, in the same time, FB1 alone, from 2500 ng·mL-1, compared to control group, show significant difference, and along with the FB1 concentration of gradually increasing, the mitochondrial membrane potential drop degree is becoming more and more obvious,15 ng·mL-1 AFB1 low-concentration combined toxicity groups and 30 ng·mL-1 AFB1 high-concentration combined toxicity groups in the mitochondrial membrane potential falling larger degree than FB1 alone.TEST 3:In this part of the test by immune protein imprinting (Western Blotting) to detect the press quantity change of apoptosis related proteins caspase-3 when FB1 and AFB1 alone and combined action。The result shows:The caspase-3 protein for 35 KD molecular weight, by sds-page electrophoresis, stamping, toxin treatment group and normal controls were in the corresponding position appeared positive brown bands, FB1 alone group, quantitative results show that with the increase of concentration, toxin caspase-3 protein also corresponding increase; When FB1 respectively with 15,30 ng·mL-1 A FB1 combine action in Vero cells, compared with single function, caspase-3 protein content has obviously increased.