Effects Of Theophylline On The Apoptosis And Expression Of Related Genes And Proteins In Rumen Mucosal Epithelial Cells Of Goats

Goat rumen epithelial cell cultivation in vitro in this study,and from the cellular and molecular level to investigate the l-theanine on weaning goat rumen mucosa epithelial cells against oxidative stress,thus to reveal l-theanine ease H₂O₂ damage goat rumen epithelium provide theoretical basis for the mechanism of action.Experiment I:Which native goat rumen epithelial cells in vitro cultivationThis experiment aims at cultivation in vitro in goat rumen epithelial cells,and immunohistochemical identification.Tests for 42 days of age which native goat rumen epithelial tissue,with 0.25%Trypsin+0.25%EDTA digestion,get a lot of primitive cells goat rumen epithelium,and carry on in vitro culture.First,by inverted microscope to observe the original generation and extend the goat rumen epithelial cells form;Second,the detection of goat rumen epithelium extend the cell growth activity,is the cell count methods;Finally,the identification of goat rumen epithelium extend the cells,selection is immune group chemical way.The results showed that:1)primitive cells goat rumen epithelium after 0.25%Trypsin+0.02%EDTA digestion,stick a wall need to 1 d time growth,is a typical"wave"rapid growth to 2 d,logarithmic period for 3 to 7 d,platform period for 7-8 d,after the recession for 8 to 9 d;2)immunocytochemistry method,according to the results of identification for the cell cytoplasm,yellowish-brown shows positive expression of cytokeratin 19?CK19 were?.The result shows that the goat rumen epithelial cells after 0.25%Trypsin+0.02%EDTA digestion,the growth activity of normal batches cells were successful,goat rumen epithelial cells for subsequent research basis for preliminary mechanism and related functions.Experiment II:Protective Effect and the Mechanism of L-theanine Against H₂O₂Induced Injury in rumen epithelial CellsThe object of this study was to explore the protective effect and the mechanism of L-theanine against rumen epithelial injury.The rumen epithelial cells injured by H₂O₂ were used as a cell model.The protective effect of L-theanine against H₂O₂-induced rumen epithelial injury was evaluated by MTT determination for cell viability,cell cycle determination,using flow cytometry for apoptosis rate and RT-PCR and Western blot for gene and protein expression of Bax,Bcl-2 and the ratio of Bax/Bcl-2.The results showed that L-theanine attenuated the cell viability loss and increase the cell rate in S phrase,and prevented rumen epithelial cells from H2O2-induced cell apoptosis.In addition,L-theanine mediated the anti-apotosis role by decreasing Bax/Bcl-2 ratio.In conclusion,L-theanine helps to protect and cure the hepatocyte injury.Experiment III:Effect of L-theanine on apoptosis and the gene expression induced by H2O2 in rumen epithelial cells of goatsThe aim of this study was to build the apoptosis model induced by H₂O₂ in subculture ruminal epithelium cells,and tested the effects of apoptosis ratio of oxidative stress induced by H₂O₂ and the L-theanine on the gene expression of caspase-3,caspase-8,FADD,apaf-1and caspase-9 in subculture ruminal epithelium cells of weaned Xiangdong black goats.Subculture ruminal epithelium cells of 42 day-old Xiangdong black goats were selected,using flow cytometry?FCM?to test apoptosis ratio of subculture ruminal epithelium cells of goats,and using reverse transcription real time polymerase chain reaction?PCR?test on the gene expression of caspase-3,caspase-8,FADD,apaf-1 and caspase-9 of subculture ruminal epithelium cells of goats.The results shows that?1?The late apoptosis of cells for group?,?and?decreased significantly?P<0.05?Compared with group?using phospholipid binding protein?annexin V?and propidium iodide?PI?unite staining;?2?The gene expression of caspase-3,caspase-9 and apaf-1 for group?,?and?decreased significantly?P<0.05?compared with group?using RT-PCR tested.The results shows that L-theanine played protection role on oxidative stress induced by H₂O₂ in subculture ruminal epithelium cells of goats,which may provide technical support and theoretical reference for the future research on oxidative stress damage mechanism of the rumen in ruminants.