The Effects of Don on Proliferation, Differentiation, and Apoptosis of Chondrocytes in Chicken
|School||Nanjing Agricultural College|
|Course||Clinical Veterinary Medicine|
|Keywords||DON chondrocyte apoptosis gene expression|
Deoxynivalenol (DON), also known as vomitoxin, is a pathogenic mycotoxins produced by Fusarium fungi. Reports have shown that it has strong cytototic, significant toxicity to prokaryotic cells and eukaryotic cells. DON may inhibit DNA, RNA and protein synthesis, and induce apoptosis. We studied the chondrocyte, to investigate the morphology; to investigate the effect of proliferation and differentiation by DON under different concentrations and different times;to indicate the apoptosis factor:Caspase-3、C-myc and P53 mRNA expression induced by DON at different concentrations. The aim of this research is to investigate the effects of DON of the proliferation, differentiation and apoptosis of chicken chondrocytes in vitro, for providing evidence of the molecular mechanism of chondrocytic toxicity induced by DON.Experiment I Culture of Chondrocyte Isolated From Embryonic Chicken and Effects of Don on the Proliferation and Differentiation of ChondrocytesObjective:To culture the chondrocyte of chicken embryos and study effects of DON on the proliferation, differentiation and apoptosis of chondrocytes. Methods:The chondrocyte was harvested by enzyme digestion from bone chips isolated from the tibia of about 17-day embryonic chicken. The growth and morphous of the cells were observed by inverted phase contrast microscope and transmission electron microscope. The induced DON were divided into 7 experimental groups and cultured respectively, containing different concentrations (10,5,2,1,0.5,0.1,0.02μg·mL-1) of DON. Cells cultured without DON as blank control group. The rate of cell proliferation was detected with methyl-thiazol-tetrazolium (MTT) colorimetry at different time points and different concentration gradient. Alkaline phosphatase (ALP) was detected with PNPP methods. Results:(1) Under the transmission electron microscope, biological feature could be observed;(2) Compared with the control group, DON groups significantly inhibited the proliferation of chondrocytes (P<0.01);(3) Compared with the control group, DON groups significantly inhibited the ALP activity of the chondrocyte (P<0.01);(4) The morphology of chicken chondrocyte was different with control group under the transmission electron microscope. Conclusions:The chondrocytes isolated from the tibia embryonic chicken were cultured successful. DON can significantly inhibit the proliferation and differentiation of the chondrocytes.ExperimentⅡEffects of DON on the Apoptosis and Oxidative Stress of Chondrocytes Cultured from the Chicken EmbryosObjective:Based upon the culture method of chondrocytes established from chicken embryos in vitro, we studied effects of DON on the apoptosis and oxidative stress of chondrocytes in embryonic chicken. Methods:Apoptosis rate was detected by the flow cytometer. Then, the chondrocyte was inoculated in 6-well plates adding the same content as above for detection of GSH, LDH and SOD. Results:(1) Compared with the control group, apoptosis rate of DON group was higher; (2) Compared with the control group, DON groups promoted the content of GSH, LDH and SOD in cell culture fluid.Conclusions:The results of flow cytometer showed that when DON was added into the cell culture fluid, the cell apoptosis stepped up, while the GSH, LDH and SOD were promoted.ExperimentⅢInfluence of DON on the Expression of genes related to Apoptosis of Chicken ChondrocytesObjective:Based upon the culture method of chondrocytes established from chicken embryos in vitro, we studied the influence of DON on the expression of Caspase-3 mRNA, C-myc mRNA and P53 mRNA. Methods:The expression of Caspase-3 mRNA, C-myc mRNA and P53 mRNA were detected using RT-PCR on chondrocytes which were treated in different concentrations of DON(20 ng·mL-1、80 ng·mL-1 and 320 ng·mL-1) and DMEM/F12 with 5% FBS in control group for 48 h. Results:Compared with control group,20,80 and 320 ng·mL-1DON groups could promote the mRNA expression of Caspase-3, showing extremely signifcant difference(P<0.01);Compared with control group,80, and 320 ng·mL-1DON groups could promote the mRNA expression of C-myc (P<0.01);Compared with control group,320 ng·mL-1DON group could promote the mRNA expression of P53 obviously (P<0.01). Conclusions:Effect of DON on induction of apoptosis in chondrocytes with involvement of gene regulation and its mitochondrial apoptosis pathway.