Research Of Gln And Its Depeptide Under Oxidative Stress On Apoptosis And Its Mechanism Of Ruminal Epithelium Cells Of Goats

This study was based on researches before and conducted to master the relationship Gln?Gly-Gln and Ala-Gln and ruminal epithelial cells apoptosis of weaned goats and investigate the influence of Gln?Gly-Gln and Ala-Gln on antioxidative capacity of wean-ed goats from cellular and molecular level, through separation technology of Primary cell, continuous cell culture models, oxidative stress model, real time quantitative PCR (RT-qPCR), immunohistochemistry techniques, in vivo test, combined with the current progress of clinical studies, which may support evidence as well as new s-tudy direction for direction for relieving oxidative stress that ruminal epithelium cells of goat had suffered.Experiment I:Characteristics Research of Liuyang Black Goat Ruminal Epithelium Cell Cycle distribution, Proliferation and Apoptosis.The present study was carried out to establish a in viro culture model of rumen epithelial cells of Liuyang black goat and preliminarily investigate the regular of apoptosis. Ruminal epithelium tissuses of 60 day-old goats were collected, and ruminal epithelium cells were digested with 0.25% trypsin and 0.02% ethylene diamine tetraacetic acid (EDTA), and obtained individual primary cultured ruminal epithelium cells were cultured in vitro. The morphosis of goat rumen epithelial cells in primary culture and subculture were observed under the inverted microscope, the growth activity curve of subcultured ruminal epithelium cells was tested by cell number count assay. Meanwhile, the identification of subcultured ruminal epithelium cells was applied to by the method of immunohisto chemistry, and through Flow Cytometry method, the subcultured ruminal epithelium cells cycle and apoptosis were determined. Results were showed as follows:1) The primary cultured ruminal epithelium cells of goats obtained by digestion of 0.25% trypsin and 0.02% EDTA began adherent growth after cultured for 1day. In the 2nd day, an exponential growth started resembling a "hill" in appearance, the cell growing rate reached the highest in the 3-4th day, stable in the 7-8th day, and decline after 8th.2) Identified by immunocytochemical method, the cytoplasm was brown, which meaned that cytokeratin 19 (CK19) expression was positive.3) Phospholipid Binding Protein (Annexin V) and Propidium Iodide (PI) unite staining showed that apoptosis of cells increased significantly with the prolongation of culture time. In conclusion, the ruminal epithelium cells of goats were obtained successfully by the method of 0.25% trypsin+0.02% EDTA digestion, which provided technical support and theoretical reference for further study on mechanism and the function of ruminant rumen.Experiment II:Building of apoptosis model induced by H2O2 in goat ruminal epithelium cellsTest using single factor experiment design, and a total of six treatments with at least three repeats, then added 4 final concentrations 0?100?400?800?M H2O2 to ruminal epithelium cells of goat in vitro. The results shows that:Compared with control group, added the concentrations of H2O2 800?M, early apoptosis of ruminal epithelium cells of goats significantly enhanced (P<0.01), but late apoptosis of ruminal epithelium cells of goats were firstly increase and then decrease with the increasing of the concentration level of H2O2, and significantly enhanced (P<0.01). This shows that apoptosis enhanced by H2O2 on ruminal epithelium cells of goats.Experiment ?:Effect of Gin?Ala-Gln and Gly-Gln on apoptosis of ruminal epithelium cells of goatTest using single factor experiment design, a total of fifteen treatments with at least three repeats, and added Gin and Ala-Gln levels (16.0 mM) and Gly-Gln levels (17.28 mM); and tested added five levels Gin, Ala-Gln and Gly-Gln (0?800(H2O2)?M? 17.28(GG)mM+800 (H202) ?M?16 (G)mM+800 (H2O2)?M?16(AG)mM+800 (H202 ?M) on apoptosis. Using Phospholipid Binding Protein (Annexin V) and Propidium Iodide (PI) unite staining showed:(1) Compared with control group, added early apoptosis ratio of cells on Gln?Gly-Gln and group significant decrease (P<0.01),and added Ala-Gln reduced 0.09%. Stated that added Gln?Gly-Gln and Ala-Gly can decrease in early apoptosis ratio of ruminal epithelium cells of goats. (2)Compared with (C (H2O2)) group, late apoptosis rate of GG+C(H2O2) group significant decrease (P<0.01), which showed that the Gly-Gln for apoptosis of ruminal epithelium cells of goats played protecion role.Experiment VI:Effect of Gln?Gly-Gln and Ala-Gln on gene expression of apoptosis of ruminal epithelium cells of goatsTest using single factor experiment design, tested added five Gin, Ala-Gln and Gly-Gln levels (0?800(H2O2) ?M?17.28(GG) mM+800(H2O2) ?M?16(G) mM+800(H2O2) ?M?16(AG) mM+800(H2O2 ?M) on apoptosis and Bcl-2/Bax mRNA expression of ruminal epithelium cells of goats. Subculture ruminal epithelium cells of 60 day-old XiangDong black goats were selected, usingreal time quantitative polymerase chain reareaction (RT-qPCR) test on apoptosis and Bcl-2/Bax gene expression of subculture ruminal epithelium cells of goats. using RT-PCR tested results showed that:(1) compared with control group, added H2O2 to maked Bcl-2 mRNA expression significant decrease (P<0.01), and Bax mRNA expression significant enhanced (P< 0.01), Bcl-2/Bax ratio significant decrease (P< 0.01); (2) On the basis of H2O2 (800ul/L), added GlnN Gly-Gln and Ala-Gln levels of, compared with (C (H2O2)) group, Bax mRNA significant decrease (P< 0.01), and Bcl-2 mRNA significant enhanced (P<0.01) and Bcl-2/Bax ratio significant enhanced (P<0.01). The results showed that:(1) H2O2 and ruminal epithelium cells of goats relationship between Bcl-2 and/or Bax;(2) Bcl-2/Bax ratioing enhanced and apoptosis rate of cells reduction after added Gin, and oxidative stress damage of ruminal epithelium cells of goats played protecion role;which may provide technical support and theoretical reference for the future research of oxidative stress damage mechanism in ruminant rumento.Experiment V:Effect of Gln?Gly-Gln and Ala-Gln on protein levels expression of apoptosis of ruminal epithelium cells of goatsTest using single factor experiment design, tested added five Gin, Ala-Gln and Gly-Gln levels (0?800(H2O2) ?M?17.28(GG)mM+800(H2O2) ?M?6(G)mM+800(H2O2) ?M? 16(AG) mM+800(H2O2?M) on apoptosis protein levels expression of ruminal epithelium cells of goats. using Western Blot tested Gln, Gly-Gln and Aln-Gln on apoptosis and Bcl-2/Bax protein levels expression of ruminal epithelium cells of goats. The results showed that:compared with control group, added H2O2 to Bcl-2 protein levels expression significant decrease (P< 0.01), and Bax protein levels expression significant enhanced (P<0.01); (2) On the basis of H2O2 (800ul/L), after Bax protein levels expression significant reduction (P<0.01) added Gln?Gly-Gln and Aln-Gln, but Bcl-2 protein levels expression significant enhanced (P< 0.01) after added Gly-Gln and Gln.(3) On the basis of H2O2 (800ul/L), Bcl-2/Bax ratioing significant enhanced (P< 0.01) after added Gln.The results shows that Gln?Gly-Gln and Ala-Gln played protection role on oxidative stress induced by H2O2 in subculture ruminal epithelium cells of goats.