Effects Of Different Time Hypoxia Training On The Thymocytes Apoptosis Of Rats

Objective:By using of different duration of hypoxia training model,Effect of the different duration exercise under the condition to hypoxia of rat thymus cells in morphology, the number of apoptotic cells, thymus cells hypoxia inducible factor (Hypoxia Induced Factor-la, HIF-la) changes in apoptosis pathway-related factor bcl-2, Bax protein and mRNA expression, hypoxia of different duration exercise on thymocyte apoptosis and its mechanism.Methods:60 SD rats were randomly divided into 6 groups of 10 each, namely:control group (A), hypoxia 8h group (B), hypoxic 12h group (C), regular aerobic exercise group (D), hypoxia 8h exercise group (E) and 12h hypoxia exercise group (F).D, E, F group every day in the animal gradient of 0 to 25m/min speed treadmill exercise 1h. After exercise, the B, E group and C,F group in turn into the oxygen concentration of 12.5%(equivalent to an altitude 4000 m) in the hypoxic chamber 8h and 12h. Lasted 4 weeks,5 days a week. The last 24 hours after the end of the experiment, rats were anesthetized intraperitoneally implementation, the ideal of whole blood taken after the eyeball were executed, and thymus tissue preparation of paraffin sections of thymus tissue, HE staining of thymus cell morphological changes; TUNEL labeling of thymus tissue were observed in the light of apoptosis index; immune staining of rat thymus cells HIF-la, B-cell lymphoma/ leukemia oncogene-2 (B cell lymphoma/leukemia 2,Bcl-2),Bax (Bel-2 associated x,Bax) factors of the protein;RT-PCR detection of rat thymus cells in HIF-la, Bcl-2, Bax in the mRNA level.Results:(1)Tissue thymosin, CD30 content:thymosin, CD30 content of A,B group,C group were compared among the three groups showed significant differences (p<0.05),group A pairwise comparison of thymus.(2) Thymus tissue in the HE staining:A group of thymus tissue structural integrity, membrane integrity, the nucleus is relatively large. Medulla and the cortex and there were no specific changes;B group than the A group of thymus cells in the thymus cells had no significant changes in the cortex when a small number of exfoliated cells;C group thymic corpuscles swelling, some edge off, shedding more visible in the cortex cells;D group significantly expanded thymic corpuscles, flat cells, cell gap increases;E group than the D group of thymic corpuscles scope and extent of degeneration were significantly smaller than and light in the D group, thymic corpuscles mild swelling; F Thymus edema and degeneration of cells to the cortex serious, can be seen in the cortex of the small number of exfoliated cells.(3) TUNEL staining:positive granules in apoptosis of thymocytes in medulla more, few parts of the cortex.Image analysis showed that the E group, F group, D group compared among the three groups, showing significant difference (p<0.05), the expression of any two F groups were higher than D group and E group, E group was higher than D group, with significant difference (p<0.05);D group, E group, F group were higher than the positive group A,B group,C group,with significant difference (P<0.05).(4)Immunohistochemical staining of rat thymus tissue HIF-la protein expression displayed:HIF-la immunohistochemical positive signal in the cytoplasm, which showed diffuse or granular distribution in the cortical part of the nucleus occasionally. Statistical analysis showed that:A group,B group,C group compared among the three groups, with significant difference (p<0.05),22 compared the expression of B group and C group were higher than A group were significant difference (p<0.05);E group,F group,D group compared three groups with significant difference (p<0.05);22 more F group was higher than D group and E group,E group than in D group,were significant differences (p<0.05);D group,E group,F group were higher than the positive group A, B group, C group, with significant difference (P<0.05).(5)Immunohistochemical staining of rat thymus tissue Bcl-2, Bax expression showed positive, bcl-2, Bax immunohistochemistry positive signal in the cytoplasm,occasionally in the nucleus. Statistical analysis: Bcl-2, bax expression showed positive group A,B group,C group compared among the three groups,with significant difference (p<0.05), Comparison of expression were lower than the B group group and C group,C group was higher than B group,with significant difference (p<0.05);D group,E group,F group were higher than the positive group A,B group,C group,with significant difference (P<0.05).(6)Thymus tissue apoptosis of rat and Bax,Bax/bcl-2 the correlation ratio and the HIF-1a and Bax expression show:rat thymus tissue cell apoptosis and Bax,Bax/Bcl-2 There was a positive correlation between the ratio (p<0.05);thymus cells HIF-1a protein expression and positive correlation between Bax (P<0.05).(7) RT-PCR detection of mRNA expression:Bcl-2,bax mRNA expression of A group, B group, C group compared among the three groups,with significant difference (p<0.05),A group of mRNA expression compared lower than the B group and C group, C group was higher than B group,with significant difference (p<0.05);D group, E group, F group mRNA expression were higher than A,B group, C group,significant differences (P<0.05);HIF-la mRNA expression of A group, B group, C group compared among the three groups, with significant difference (p<0.05), compared the expression of B group and C group were higher than group A comparison, there were significant differences (p<0.05);D group, E group, F group were higher than the mRNA expression of A group, B group, C group, with significant difference (P<0.05).Conclusion:1.Hypoxic exposure on exercise for thymosin thymus size and CD30 levels increased, but when the duration of hypoxia, the content does not rise instead of decrease.2.Hypoxic and/or exercise induced thymus apoptosis, more than in medulla, cortex rare, and the original role of hypoxia as a stress intensity 8h modest.3.Hypoxic and/or exercise induced thymic apoptosis factor Bax, bcl-2 protein expression, and mRNA and protein expression of the same.4. Hypoxic and/or exercise induced HIF-la protein expression increased, but mRNA and protein expression of inconsistency.5.Bax and bcl-2 apoptosis of thymus cells, and HIF-la may be synergistic bcl-2 family involved in apoptosis regulation. As the hypoxia time,the expression of pro-apoptotic factor Bax to accelerate, accelerate thymocyte apoptosis.