| Objective:To observe changes of T lymphocytes CD4+,CD25+,Foxp3+ in rat when the ambient temperature has a sudden change, explore the effect of extreme temperature on immuse system, then provide new ideas and theory evidences for the infectious and immune related desease.Methods:1,252 male SD rats at the age of 9-15 weeks, weight 198-378g (275.24g±35.73g) were selected into manmade climate incubator to make cold stress and hot stress animal model.2,Rats were randomized into four groups, normal temperature (20℃), sudden drop in temperature to 4℃, sudden drop in temperature to -12℃and sudden rise in temperature to 37℃. Every group has three stimulation:Con-A, LPS,Blank,each every has seven time points, 1h,4h,8h,12h,24h,48h and 7d, three rats for each time point.3,Chloralic hydras intraperitoneal injection after every time point, collect Carotid artery blood into EDTA Anticoagulation tube, then centrifuge to collect blood plasm.4,PE-CD25+PE-Cy5-CD4+FITC-Foxp3 anti-rat monoclonal antibody immunocyto chemistry mark T lymphocytes CD4+,CD25+,Foxp3+.5,Use flow cytometer detect more 30000 cell, count CD4+,CD4+CD25+ CD4+CD25+Foxp3+ positive rate6,The result were compared by SPSS 17.0, presentated by X±SD, and use ANOVA to do analysis of variance, LSD to T-test。Result:1,CD4+:(1),In condition of no stimulation, the express of CD4+ has big change at different time point and different temperature, among 8h,12h,48h,168h different temperature has significant difference (P<0.05), among 20℃,-12℃,37℃different time point has significant difference (P<0.05)。among 37℃,4℃,-12℃,4h,48h was lower to normal control group, especially at 4h.(2),In condition of LPS stimulation, the express of CD4+ has big change at different time point and different temperature, among 8h,168h different temperature has significant difference (P<0.05), among 4℃,-12℃,37℃different time point has significant difference(P<0.05)。In-12℃group,1h,168h group was higher then normal control group, in 37℃group,4h,48h group was lower to normal control group.(3),In condition of Con-A stimulation, the express of CD4+has big change at different time point and different temperature, among 24h different temperature has significant difference (P<0.05), among 20℃,-12癈different time point has significant difference (P<0.05)。among 4℃,-12℃group,4h,168h group was higher then normal control gro刺up.(4),In condition of the same temperature and time point, the express of CD4+at different stimulation;①.In 20℃group,among 8h,24h,48h time point,Con-A and LPS stimulationgroup was lower then normal control group (P<0.05), among 1h,12h,168h time point Con-A and LPS stimulation group was higher then normal control group (P<0.05), especially in Con-A stimulation group.②.In 4℃group, among 1h,4h,8h,168h time point Con-A and LPS stimulation group was higher then normal control group (P<0.05), but among 12h,24h,48h time point Con-A and LPS stimulation group was lower then nomal control group.③.In-12℃group,except 48h time point group, Con-A stimulation group were all lower then nomal control group, but amount 4h,24h,168h time point group LPS stimulation group was lower then nomal control group.④.In 37℃group, among 1h,48h time point group, LPS stimulation group was was lower then nomal control group.2,CD4+CD25+:(1),In condition of no stimulation, the express of CD4+CD25+has big change at different time point and different temperature, among 1h,12h,168h different temperature has significant difference (P<0.05), among-12℃,37℃different time point has significant difference (P<0.05),in 4℃,1h,12h,24h,48h time point group was lower to normal control group, in-12℃,12h,168h time point group was lower to normal control group, in 37℃1h,48h, time point group was lower to normal control group especially at 48h.(2),In condition of LPS stimulation, the express of CD4+ CD25+ has big change at different time point and different temperature, among 1h,4h,8h,168h different temperature has significant difference (P<0.05), among 4℃,12℃,37℃different time point has significant difference (P<0.05)。In 4℃group,4h,12h,24h,48h group was lower then normal control group, in-12℃group,12h,168h group was lower to normal control group, in 37℃group,1h,48h was lower to normal control group, especially at 48h.(3),In condition of Con-A stimulation, the express of CD4+ CD25+ has big change at different time point and different temperature, among 1h,8h,24h,168h different temperature has significant difference (P<0.05),among 37℃,-12℃different time point has significant difference (P<0.05),among-12℃group,12h,48h,168h group was lower then normal control group,among 37℃,1h,48h group was lower then normal control group.(4),In condition of the same temperature and time point, the express of CD4+ CD25+ at different stimulation:①.In 20℃group,among 1h,24h time point,Con-A stimulation group was lower then normal control group (P<0.05), among 1h,12h,168h time point LPS stimulation group was higher then normal control group (P<0.05), especially in Con-A stimulation group.②.In 4℃group, among 8h,168h time point Con-A and LPS stimulation group was higher then normal control group (P<0.05), but among 4h,8h,24h time point Con-A stimulation group was lower then nomal control group.③.In-12℃group, among 4h,12h time point group, Con-A and LPS stimulation group has significant difference (P<0.05), but amount 12h,24h time point group Con-A and LPS stimulation group was lower then nomal control group.④.In 37℃group, among 48h,168h time point group, LPS stimulation group was was higher then nomal control group. 3,CD4+CD25+Foxp3+:(1),In condition of no stimulation, the express of CD4+CD25+Foxp3+has big change at different time point and different temperature, among 4h,12h,24h,168h different temperature has significant difference (P<0.05), among -12℃,37℃different time point has significant difference (P<0.05),in 4℃,1h,12h time point group was higher to normal control group,4h,8h,24h,48h,168h time point was lower then normal control group, in -12℃,168h time point group was lower to normal control group, in 37℃,4h,8h,48h,168h time point group was lower to normal control group,1h,12h,24h was higher then normal control group.(2),In condition of LPS stimulation, the express of CD4+CD25+Foxp3+has big change at different time point and different temperature, among 168h different temperature has significant difference (P<0.05), among20℃,12℃,37℃different time point has significant difference (P<0.05)。In 4℃group,1h,168h group was lower then normal control group, in -12℃group,4h,24h group was higher to normal control group, 1h,8h,12h,48h,168h group was lower to normal control group,in 37℃group,4h,12h,24h group was higher to normal control group, 1h,8h,48h,168h group was lower then normal control group.(3),In condition of Con-A stimulation, the express of CD4+CD25+Foxp3+has big change at different time point and different temperature, among 8h,12h different temperature has significant difference (P<0.05),among 37℃,-12℃different time point has significant difference (P<0.05),among 4℃group,1h,168h group was lower then normal control group,among -12℃, 1h,168h group was lower then normal control group.among 37℃,12h group was higher then normal control group, 1h,4h,8h,48h,168h group was lower then normal control group.(4),In condition of the same temperature and time point, the express of CD4+CD25+Foxp3+ at different stimulation:①.In 20℃group,among 12h,24h time point,Con-A and LPS stimulation group has significant difference (P<0.05), among 8h,12h,24h,48h time point Con-A stimulation group was higher then normal control group (P<0.05), especially in Con-A stimulation group.②.In 4℃group, among 4h,8h time point Con-A and LPS stimulation group was higher then normal control group (P<0.05), among 24h,48h time point Con-A stimulation group was higher then normal control group, among 24h,48h,168h LPS stimulation group was higher then nomal control group.③.In-12℃group, among 1h,48h time point group, Con-A and LPS stimulation group has significant difference (P<0.05), but amount 1,48h time point group Con-A stimulation group was higher then normal control group,24,48h time point group LPS stimulation group was lower then normal control group.④.In 37℃group, Con-A and LPS stimulation group has significant difference (P<0.05) among 4h,48h time point group, Con-A stimulation group was was higher then nomal control group.Conclusion:1. In no stimulation,168h, extreme temperature (-12℃,37℃) all up-regulate CD4+, extreme temperature (-12℃,37℃) all down-regulate CD4+CD25+Foxp3+2. In extreme temperature (-12℃,37℃), Con-A stimulation suppress CD4+3. In extreme temperature (-12℃,37℃), LPS stimulation suppress CD4+CD25+Foxp3+, in low temperature (-12℃), Con-A stimulation enhance CD4+CD25+Foxp3+, when in high temperature Con-A stimulation enhance at first and then suppress CD4+CD25+Foxp3+. |
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