【作者】 戴新贵；

【导师】 艾宇航； 姚咏明；

【作者基本信息】 中南大学 ， 麻醉学， 2008， 硕士

【摘要】 目的:1:探讨脓毒症CD4~+CD25~+Treg凋亡对CD4~+效应T细胞免疫功能的影响及血必净注射液的干预作用。方法:1)体外实验:断颈处死Wister大鼠,无菌取脾脏,分离单个核细胞。1:免疫磁珠法(MACS)分离正常大鼠脾脏CD4~+T细胞、CD4~+CD25~-T细胞、及CD4~+CD25~+Treg,以异硫氰酸荧光素(FITC)标记CD25,流式细胞术(FCM)鉴定细胞纯度,0.4%台盼蓝染色检测细胞存活率。2:将体外分离的CD4~+CD25~+Treg分为对照组、抗-CD3/CD28刺激组、抗-CD3/CD28+脂多糖(LPS)刺激组、抗-CD3/CD28+LPS+血必净组(5mg/ml)、抗-CD3/CD28+血必净组,于第3天以别藻蓝蛋白(APC)标记Annexin-V及放线菌素D(7-AAD)标记Treg,FCM检测细胞的凋亡率;藻红蛋白荧光素(PE)标记表面标志物叉头翼状螺旋转录因子(Foxp3)和T淋巴细胞毒性相关抗原4(CTLA-4),FCM检测Foxp3和CTLA-4平均荧光强度;酶联免疫吸附试验(ELISA)检测IL-10分泌情况。3:将各组Treg分别与CD4~+CD25T细胞1:1共培养,刀豆素(ConA)刺激68h,噻唑蓝(MTT)比色分析法检测T细胞增殖、ELISA法检测白介素2(IL-2)和可溶性IL-2受体(sIL-2R)及辅助性T细胞(Th)漂移因子干扰素γ(INFγ)/IL-4/IL-17的分泌。2)体内实验:复制盲肠结扎并穿孔(CLP)大鼠脓毒症模型,体外提取脾脏CD4~+CD25~+Treg培养过夜。1:将大鼠分为对照组、假手术组、CLP组,血必净组(4mg/kg),于第3天检测FOXP3、CTLA-4、凋亡率、IL-10。2:将各组CD4~+CD25~+Treg与CD4~+CD25~-T细胞1:1共培养,ConA刺激68h,检测T细胞增殖、IL-2/sIL-2R、INFγ/IL-4/IL-17。结果:1)体外实验:1:Treg凋亡:对照组凋亡率各时间点之间比较无统计学意义(P＞0.05);抗-CD3/CD28单克隆抗体诱导Treg凋亡率明显高于同时间对照组(P＜0.01),达25-40%。Foxp3、CTLA-4与凋亡率呈负相关;抗-CD3/CD28+LPS刺激组凋亡率高于同时间对照组(P＜0.01),稍低于抗-CD3/CD28刺激组,但无统计学意义(P＞0.05),而Foxp3、CTLA-4与对照组比较并不随凋亡率增加而降低。2:对效应T细胞免疫功能的影响:①T细胞增殖:正常Treg能明显抑制Teff增殖功能,抗-CD3/CD28组和anti-CD3/CD28+血必净组明显高于对照组(P＜0.05),抗-CD3/CD28+LPS较对照组明显降低(P＜0.05),抗-CD3/CD28+LPS+血必净组与抗-CD3/CD28+LPS组比较有明显差异(P＜0.01)。②IL-2/sIL-2Ra的分泌:IL-2:抗-CD3/CD28刺激组较对照组明显升高(P＜0.01);抗-CD3/CD28+LPS刺激组明显低于对照组和抗-CD3/CD28刺激组(P＜0.01);抗-CD3/CD28+LPS+血必净刺激组较对照组、抗-CD3/CD28刺激组、抗-CD3/CD28+血必净刺激组明显降低(P＜0.05或P＜0.01),而较抗-CD3/CD28+LPS组升高(P＜0.01)。sIL-2Ra:抗-CD3/CD28+LPS组较抗-CD3/CD28组有明显升高,抗-CD3/CD28+LPS+血必净组较抗-CD3/28+血必净组有明显升高。③Th1/Th2/Th17(IFNγ/IL-4/IL-17)漂移:INFγ和IL-4随凋亡增加而升高,抗-CD3/CD28+LPS+血必净组INFγ分泌明显高于抗-CD3/CD28+LPS组(P＜0.05),而IL-4则呈相反变化(P＜0.05),使IFNγ/IL-4升高P＜0.05);抗-CD3/CD28+血必净组IL-17较抗-CD3/CD28组有明显降低(P＜0.05)。2)体内实验:1:Treg凋亡:结果表明对照组Treg凋亡率为12.03±0.89%,假手术组(9.48±2.17%)和对照组比较无明显差异,CLP组凋亡率(5.87±0.44%)低于对照组(P＜0.05),而血必净治疗组明显高于其他各组(P＜0.01)。Foxp3、CTLA-4表达和IL-10分泌功能随凋亡率增加而减少,成负相关。2:对效应T细胞免疫功能的影响:①T细胞增殖:MTT检测Teff增殖显示CLP组抑制率明显高于对照组(P＜0.01),而血必净组抑制率低于CLP组和对照组(P＜0.01)。②IL-2/sIL-2Ra的分泌:CLP组IL-2分泌低于对照组(P＜0.05),sIL-2Ra高于对照组(P＜0.05);血必净组IL-2分泌高于CLP组(P＜0.05),sIL-2Ra低于CLP组(P＜0.01)。③Th1/Th2/Th17(IFNγ/IL-4/IL-17)漂移:CLP组INFγ、IL-4和IFNγ/IL-4与对照组比较有明显升高(P＜0.05);而血必净治疗组IFNγ、IL-4、IFNγ/IL-4明显高于CLP组(P＜0.05);各组IL-17无明显变化。结论:1:抗-CD3/CD28能有效诱导脓毒症Treg凋亡,Treg凋亡后细胞接触机制和分泌功能下降。2:脓毒症Treg对效应T细胞具有强力免疫抑制功能,血必净注射液促进Treg凋亡后可有效改善其对效应T细胞的细胞免疫抑制作用。更多还原

【Abstract】 Objective:To observe the effect of apoptosis of CD4⁺CD25⁺ regulatory T cells in septic rats on immunological function of CD4⁺ T tymphocytes induced by Xuebijing injectionMethods:1):In vitro experiment:To separate the mononuclear cells of the spleen of rats asepsisly after sacrificed.1:CD4⁺T cells, CD4⁺CD25⁺ Treg and CD4⁺CD25 T cells were separated by immunomagnetic bead isolate system,CD25 was marked by fluorescein isothiocyante（FITC）.The cell purity of CD4⁺CD25⁺ Treg was detected by 0.4%trypanblau and the survival rate was analyzed by Flow cytometry（FCM）.2:The CD4⁺CD25⁺ Treg were divided into the control group,anti-CD3/CD28 stimulation group,anti-CD3/CD28+ lipopolysaccharide（LPS）stimulation group and anti-CD3/CD28+ LPS+Xuebijing stimulation groups（5mg/ml）group.The apoptotic rates of CD4⁺CD25⁺ Treg were analyzed by FCM used allophycocyanin （APC）-Annexin and 7-amino-actinomycin-D（7-AAD）at 3^thd.The geometric mean of fluorescence intensity of phycoerythrin（PE）-forkhead/winged helix transcription factor（Foxp3）and PE-cytotoxic T lymphocyte-associated antigen 4（CTLA-4）of CD4⁺CD25⁺ Treg was analysised by FCM.The interleukin（IL）-10 serected from CD4⁺CD25⁺ Treg was measured by enzyme-linked immunoadsorent assay（ELISA）.3: Each group CD4⁺CD25⁺ Treg were co-cultured with CD4⁺CD25- T cells（1:1）,T cell proliferation experiment was done by colorimetric method used thiazolyl blue（MTT）.IL-2/sIL-2R,INFγ/IL-4/IL-17 were measured by ELISA.2):In vivo experiment:Replicated the cecal ligation and puncture（CLP）animal models,and seperated the.CD4⁺CD25⁺ Treg in vitro and cultured 24h.1:Rats were divilded into the control group, sham-operated group,CLP group and Xuebijing treatment group.Foxp3, CTLA-4,IL-10,apoptotic rates were tested by the above methods.2: Seperated CD4⁺CD25⁺ Treg cells,co-cultered with CD4⁺ T cells（1:1）, MTT rooliferation experiment,IL-2/sIL-2R,INFγ/IL-4/IL-17,were measured.Results:1).In vitro experment 1:Apoptotic rate,empression of Foxp3 and CTLA-4 and secretion of IL-10:the apoptotic rate of the anti-CD3/CD28 group（25-40%）was higher than the control group obviously（P＜0.01）.The mean fluorescence intensity of Foxp3 and CTLA-4 and secretion of IL-10were negatively correlated with the apoptosis rates.The apoptotic rate of anti-CD3/CD28 group was higher than the control group（P＜0.01）.The group of anti-CD3/CD28 stimulated had the highest apoptotic rate,which had a obvious significance with other groups（P＜0.05 or P＜0.01）.And the group of anti-CD3/ CD28+LPS+Xuebijing group was lower than the group of anti-CD3/CD28+LPS group.2:Effect on immunological function of CD4⁺ T lymphocytes①MTT rooliferation experiment:compared with control group,the Teff proliferative activity in response to ConA of septic group were significantly suppressed（P＜0.01）,and compared with anti-CD3/CD28+LPS group,proliferative activity in anti-CD3/ CD28+LPS+Xuebijing group has a significantly improve（P＜0.01）.②secretion of IL-2/slL-2Ra:IL-2:compared with control group,the level of IL-2 in the supermatant of septic group were significantly suppressed （P＜0.01）,and compared with anti-CD3/CD28+LPS group,secretion of IL-2 in anti-CD3/CD28+LPS+Xuebijing group has a significantly improve（P＜0.01）,sIL-2Ra:The secretion of sIL-2Ra of anti-CD3/ CD28+LPS group and anti-CD3/CD28+LPS+ Xuebjijing group had no significantly change,but both were higher than the control groups（P＜0.01）.③Drift of Thl/Th2/Th17（IFNγ/IL-4/ IL-17）:INFγsecreted in antiCD3/CD28+LPS+Xuebijing groups more than anti-CD3/CD28+LPS group（P＜0.05）,IL-4 had an opposite tendency with INFγ.In anti-CD3/CD28+ Xuebijing injection group,IL-17 had significantly decreased compared with the group of anti-CD3/CD28（P＜0.05）.2).In vivo experment 1:Apoptotic rate,empression of Foxp3 and CTLA-4 and secretion of IL-10:comperated with the sham-operated group,all indexs have no change with the control group（P＜0.05）.In CLP group,the apoptotic rate was lower than the control group（P＜0.01）,the empression of Foxp3 and CTLA-4 and the secretion of IL-10 were higher than the control group（P＜0.01）.2:Effect on immunological function of CD4⁺ T lymphocytes①MTT rooliferation experiment:the Teff proliferative activity in response to ConA in CLP rats were significantly suppressed compared with control group（P＜0.01）,The inhibition rate of Teff proliferative activity of Xuebijing treatment group was lower than CLP group,the secretion of IL-2 was higher than CLP groups.②secretion of IL-2/sIL-2Ra:the level of IL-2 of Teff in CLP rats were significantly suppressed compared with control group（P＜0.01）,and secretion of sIL-2Ra in the supermatant was higher than the control groups.The level of sIL-2Ra of Xuebijing treatment group was lower than CLP group,the secretion of IL-2 was higher than CLP groups.③Drift of Th1/Th2/Th17 （IFNγ/IL-4/IL-17）:their secretion of INFγ,IL-4 and ratio of INFγ/IL-4 had significantly increased compared with the control group（P＜0.05）,and INFγ,IL-4,INFγ/IL-4 higher than the CLP group（P＜0.05）.The secretion of IL-17 in each group was no change.Conclusion:1.CD4⁺CD25⁺ Treg shows apoptosis induced by anti-CD3/CD28 and the empression of Foxp3 and CTLA-4 and the secretion of IL-10 downregulated with the increase of apoptotic rate. 2.CD4⁺CD25⁺ regulatory T cells upregulated the suppression function on Teff in sepsis,and Xuebijing injection could down-regulate the suppression function effectively via reducing apoptosis of septic Treg.更多还原