| The cell shape,DNA damage, activity of Caspase-3, expression level of Bcl-2,p53 andCaspase-3 mRNA in splenic lymphocytes of the chickens with Vitamin E (VE)deficiency whichproduced by using low content VE feedstuff and supplying unsaturated fatty acid in the diet weredetected through semi-quantitative RT-PCR, flow cytometry, alky SCGE,analysis of DNAgragmentation, analysis of DNA Ladder and observation of shape to study the mechanism ofapoptosis of splenic lymphocytes in chickens with VE deficiency. The results showed:1. It observed that nucleus of splenic lymphocytes dyed tightly and enriched, some part ofnucleole vanished, cell membrance integrated and presented apoptosis morphology diversity underlight microscope in VE deficiency group.It also observed that nuclear chromatin was enriched tokaryolemma, kytoplasm condensed, cell organelle gathered, electron densed, cell nucleuspyknosised, cell-cell junction disappeared and apoptotic body appeared under transmission electronmicroscope. The cell showed salmon pink in VE deficiency chickens under fluorescence microscopeby AO/EB double fluorescence coloration. It indicated that splenic lymphocytes in VE deficiencychickens presented apoptosis.2. Flow cytometry assay was used to detect apoptosis, and the apoptotic peak appeared beforeG0/G1 peak, proliferation index cut down, apoptosis rate step up in chickens with VE deficiency, thedifference was significantly(p＜0.01)contrasted to control group. It showed that the proliferation ofsplenic lymphocytes was inhibited and cell population of apoptosis increased in chickens with VEdeficiency.3. The cell in chickens with VE deficiency showed the phenomenon of comet tailing wasdetected by alky SCGE.The degree of DNA gragmentation was significantly higher in VEdeficiency group than the control one(p＜0.01).The VE deficiency group showed typal DNAladder-shaped by analysis of DNA Ladder.It indicated that DNA of splenic lymphocytes inchickens with VE deficiency were damaged.4. The activity of Caspase-3 was detected by Caspase-3 spectrophotometry Assay Kit. Itrevealed that the activity of Caspase-3 was significantly higher in VE deficiency group than thecontrol one(p＜0.01). It indicated that VE deficiency lead to apoptosis of splenic lymphocytes inchickens by activating Caspase-3.5. The expression level of inhibiting-apoptosis gene of Bcl-2 mRNA was lower and thepromoting-apoptosis gene p53 and Caspase-3 mRNA were higher in VE deficiency group than thecontrol one(p＜0.01). It indicated that VE deficiency lead to splenic lymphocytes apoptosis inchickens through interfering the expression of apoptosis correlated gene. This research elucidated the mechanism of apoptosis of splenic lymphocytes in chickens with VEdeficiency on the molecular level from the aspects of apoptosis, DNA damage, enzyme activity andthe expression of apoptosis gene. It offered theory about the mechanism of immunocell damage andimmunosuppression in chickens with VE deficiency. |
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