自然调节性T细胞通过修饰前炎症应答介导脑型疟疾的发生

发布时间：2018-01-12 00:01

本文关键词：自然调节性T细胞通过修饰前炎症应答介导脑型疟疾的发生　出处：《中国医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 实验目的疟疾感染是当今威胁人类健康的主要难题之一,每年有一百多万儿童死于疟疾。脑型疟疾(cerebral malaria, CM),简称脑疟,是疟疾最严重的并发症之一,而且是疟疾感染的主要死因。CM严重性的决定因素尚不完全清楚,但很可能来自宿主和寄生虫两方面。最终,寄生虫和免疫应答的相互作用决定宿主感染结局。因此,关于CM发生免疫机制的基础研究是研制有效抗疟疫苗和药物的前提。伯氏疟原虫(Plasmodium berghei ANKA,P.bANKA)鼠疟与人类疾病有许多共同特征,是现有CM的最佳模型。目前研究认为CM是由脑血管内感染红细胞(parasitized red blood cells,pRBC)粘附和细胞因子及效应细胞介导的强烈的宿主前炎症应答的结合效应引起的疾病。相关研究表明活化T细胞介导的免疫应答参与疾病诱导,并且CD4+T细胞和CD8+T细胞均可促进脑病变发生。事实上,Thl应答在控制虫血症方面发挥关键作用,同时也促进CM发生。 CD4+CD25+调节性T细胞(regulatory T cells, Tregs)是不同于Thl和Th2的具有免疫调节效应的T细胞亚群。Tregs活化是寄生虫逃避宿主免疫的机制之一前期试验已证明Tregs活化与约氏疟原虫(Plasmodium yoelii 17XL, P.yoelii 17XL)感染鼠的易感性相关,Tregs可通过修饰树突状细胞(Dendritic cells, DCs)应答,诱导IL-10产生和CD4+T细胞凋亡调控Th1应答。为此本研究动态观察了正常感染鼠及Tregs消除鼠前炎性细胞因子,抗炎性细胞因子表达水平和Tregs增殖变化,以期阐明Tregs在脑疟发生中的作用地位及其相关机制。实验方法 1、实验动物模型构建及CM病变评估 6-8w,雌性C57BL/6经腹腔感染1×106 P.berghei ANKA寄生的红细胞(pRBC)构建CM易感模型,感染BALB/c和DBA/2鼠构建CM抵抗模型。实验组(6只/组)和对照组(6只/组)小鼠于感染前1d和感染后1d分别经腹腔注射1mganti-CD25mAb (7D4,rat IgM; Bio Express)和等体积phosphate-buffered saline (PBS)构建Tregs消除小鼠模型。感染后6天起每日监测鼠两次,评价临床实验脑疟(Experimental cerebral malaria, ECM)症状,用以下症状确定ECM得分：皮毛竖起、颤抖、肢体瘫痪、痉挛、昏迷。每出现一个症状给予1分,累计得分≥4分者考虑为重型疟疾。 2、流式细胞术检测脾细胞悬液中CD4+T细胞群内CD4+CD25+Foxp3+Tregs的百分含量分别于感染前和感染后第3d、5d、8d常规无菌取出小鼠脾脏,用0.17mol/LNH4C1裂解红细胞,以含10%胎牛血清(Fetal calf serum, FCS)的RPMI1640调整脾细胞终浓度为1×107/ml。每份样品用FITC-conjugated anti-mouse CD4/L3T4 (GK1.5; BD Pharmingen), APC-conjugated anti-mouse CD25/IL-2 receptor alpha (PC61; BD Pharmingen)和PE-conjugated anti-Foxp3 (clone FJK16s; eBioscience)进行三色分析,另设阴性对照管。在流式细胞仪专用染色管中加入新鲜制备的1×107/ml脾细胞悬液0.1ml,并用FcγⅢ/Ⅱ封闭抗体孵育以阻断荧光抗体的非特异染色,再加入抗CD4-FITC和抗CD25-APC mAb进行表面染色,固定透膜后,用抗Foxp3-PE mAb进行胞浆内染色,用含1%FCS的PBS洗涤两次并悬浮于500μlPBS中,流式细胞仪(Becton Dickinson, USA)进行检测。以前向和侧向散射角确定淋巴细胞群,CD4+T细胞画门,分析计算CD4+T细胞群内CD4+CD25+Foxp3+Tregs的百分率。 3、采用双抗体夹心ELISA法检测脾细胞培养上清IFN-γ、TNF-α、IL-6、IL-17、IL-10及Griess方法检测NO2-含量 (1)脾细胞样品制备分别于感染前和感染后第3d，5d，，8d常规无菌取出小鼠脾脏,用0.17mol/L NH4Cl裂解红细胞。以含10%FCS的RPMI1640调整脾细胞终浓度为1×107/ml，于24孔培养板中,每孔加入500μl脾细胞悬液于37℃,5%CO2条件下培养48h。需要注意的是天然脾细胞与纯化NA/LE hamster anti-mouse CD3e (145-2C11, BD Pharmingen,0.56μl/well)和纯化NA/LE hamster anti-mouse CD28 (37.51, BD Pharmingen,0.112μl/well)于37℃,5%CO2条件下共同培养48h以刺激IL-17产生,随后收集培养上清,-80℃保存,待细胞因子及NO检测。 (2)双抗体夹心ELISA试剂盒分别检测脾细胞培养上清中IFN-γ、TNF-α、IL-6、IL-10、IL-17的分泌水平,酶标仪测定450nm处OD值。结果以试剂盒提供的标准品绘制标准曲线,应用SoftMax Pro 4.3.1Ls软件分析,计算细胞因子含量(pg/ml)。 (3) Griess方法检测脾细胞培养上清中NO2-含量100μl上清和Griess反应液100μl室温反应10min,用NaNO2作为标准品,酶标仪检测550nm处OD值。实验结果 1、感染率、幸存率与疾病评估脑疟是通过给C57BL/6小鼠注射毒性P.bANKA血液阶段寄生虫诱导的,C57BL/6小鼠于感染后8-11天死于麻痹和昏迷,虫血症为10-20%。相比而言,抵抗型BALB/c和DBA/2小鼠经历类似的寄生虫生长但不发生脑病变,于感染后3-4w死于贫血和过度虫血症(60%)。从脑疟症状的临床得分来看,C57BL/6小鼠得分高于BALB/c和DBA/2小鼠。 2、易感和抵抗小鼠感染期间脾细胞培养上清前炎性细胞因子、抗炎性细胞因子和NO水平的动态检测 CM易感C57BL/6鼠和抵抗BALB/c与DBA/2鼠脾细胞培养上清前炎性细胞因子IFN-γ、TNF-α、IL-6、IL-17及NO于感染后开始升高,感染后5天达峰值,感染后8天下降(C57BL/6鼠呈现轻微下降)。三种鼠脾细胞培养上清抗炎性细胞因子IL-10也于感染后5天达峰值,在感染后8天C57BL/6鼠IL-10水平显著下降,抵抗BALB/c与DBA/2鼠IL-10水平轻微下降。易感鼠脾细胞培养上清前炎性细胞因子和NO水平显著高于抵抗鼠,而IL-10水平显著低于抵抗鼠。 3、易感和抵抗小鼠感染后不同时间点脾CD4+T细胞中Tregs的百分比和绝对数 CM易感鼠和抵抗鼠脾脏CD4+T细胞中Tregs百分比和绝对数于感染后开始升高,感染后3天达峰值,感染后5天到8天逐渐下降。并且在感染期间,C57BL/6鼠脾脏CD4+T细胞中Tregs百分比和绝对数显著低于BALB/c和DBA/2小鼠。 4、体内CD25消除后Tregs效应分析 CD25消除的C57BL/6、BALB/c和DBA/2小鼠脾脏CD4+T细胞中Tregs百分数显著低于相应对照鼠。与正常感染鼠相比,Tregs消除的C57BL/6鼠幸存率明显增加,感染后5、6、7天感染率显著下降,CM症状的临床得分也相应减少,CD25消除的C57BL/6鼠不发生脑疟症状,于感染后3-4周死于贫血和过度虫血症。CD25消除的BALB/c和DBA/2鼠与正常感染鼠感染过程相似,但在感染初期虫血症得到暂时控制,CD25消除后8天Tregs'快速恢复,抑制T细胞活化,不能控制虫血症,这些鼠于感染后3-4周死于过度虫血症和严重贫血。CD25消除明显逆转IFN-γ、TNF-α、IL-6、IL-17、IL-10及NO产生。CD25消除的DBA/2小鼠与CD25消除的BALB/c小鼠的感染进程和细胞因子产生过程相似。 5、易感和抵抗鼠感染后不同时间Tregs与IFN-γ、TNF-α、NO及IL-10与虫血症的相关性分析脑疟易感鼠和抵抗鼠感染期间Tregs数量与IFN-γ、TNF-α、NO产生呈负相关,IL-10产生与虫体血症水平正相关。结论 1、前炎症应答和抗炎症应答之间平衡的设立对控制重型疟疾发生至关重要,Tregs是维持此平衡的重要调节因子。 2、Tregs通过修饰前炎症应答从而调控前炎症应答与抗炎症应答之间的平衡以介导CM的发生和感染结局。
[Abstract]:Experimental purpose
Malaria is one of the main challenges threatening human health nowadays, about one million children died of malaria each year. Cerebral malaria (cerebral malaria, CM), referred to as cerebral malaria is one of the most serious complications of malaria, and is the decisive factor of the main cause of death.CM malaria infection severity is not completely understood, but is likely to come from parasite and host two aspects. Finally, the interaction of parasites and immune response in determining host infection outcome. Therefore, research on the immune mechanism of CM is a prerequisite for the development of effective anti malaria vaccines and drugs.
Plasmodium berghei (Plasmodium berghei ANKA, P.bANKA) rodent malaria has many common features with human diseases, is the best model of the existing CM. The present study suggests that CM is composed of red blood cells in cerebral vessel infection (parasitized red blood cells, pRBC) caused a strong binding effect of host pro-inflammatory response and cytokines and cell adhesion mediated effect the illness. The research results show that activation of T cell mediated immune response in disease induced by CD4+T cells and CD8+T cells, and can promote brain lesions. In fact, Thl responses play a key role in the control of parasitemia, but also promote the occurrence of CM.
CD4+CD25+ regulatory T cells (regulatory T cells, Tregs) is different from that of Thl and Th2 have immunomodulatory effects of T cell subsets in the activation of.Tregs is one of the mechanisms of parasite escaping from host immune preliminary experiments have proved that the activation of Tregs and Plasmodium yoelii (Plasmodium yoelii 17XL, P.yoelii 17XL) were associated with susceptibility to infection, Tregs the modified dendritic cells (Dendritic cells DCs) response, inducing the production of IL-10 and CD4+T apoptosis Th1 response. The purpose of this study was observed in normal rats and Tregs rats before infection elimination of inflammatory cytokine levels, the proliferation of Tregs and expression of inflammatory cytokines, to elucidate the role of Tregs in cerebral malaria incidence and the related mechanism.
Experimental method
1, experimental animal model construction and CM lesion evaluation
6-8W, female C57BL/6 intraperitoneal infection of 1 x 106 P.berghei ANKA parasitized erythrocytes (pRBC) to construct CM susceptibility model, BALB/c infection and DBA/2 rats to construct CM resistance model. The experimental group (6 / group) and control group (6 / group) in mice before infection of 1D and 1D after infection were intraperitoneally injection of 1mganti-CD25mAb (7D4, rat IgM; Bio Express) and phosphate-buffered saline (PBS) to construct the volume Tregs elimination mouse model. 6 days after infection were two times daily monitoring, evaluation of clinical experimental cerebral malaria (Experimental cerebral, malaria, ECM) to determine the symptoms, ECM score with the following symptoms: Fur ruffled, trembling limbs paralysis, convulsions, coma. Every symptom score of 1, the cumulative score more than 4 points were considered as severe malaria.
2, the content of CD4+CD25+Foxp3+Tregs in the CD4+T cell group in the splenic cell suspension by flow cytometry
The infection before and after infection respectively in 3D, 5D, 8D of conventional asepsis spleen of mice with 0.17mol/LNH4C1 lysis of red blood cells, containing 10% fetal bovine serum (Fetal calf, serum, FCS) RPMI1640 adjusting spleen cells at the concentration of 1 * 107/ml. per sample with FITC-conjugated anti-mouse CD4/L3T4 (GK1.5; BD Pharmingen, APC-conjugated anti-mouse) CD25/IL-2 receptor alpha (PC61; BD Pharmingen) and PE-conjugated anti-Foxp3 (clone FJK16s; eBioscience) of color analysis, the negative control. Adding freshly prepared 1 * 107/ml spleen cells in flow cytometry for staining tube suspension 0.1ml and Fc gamma III / II blocking antibody incubation to block non specific fluorescent antibody staining with anti CD4-FITC and anti CD25-APC mAb surface staining, fixed membrane permeability, anti Foxp3-PE mAb in the cytoplasm stained with 1%FCS PBS and washed two times Becton Dickinson (USA) was suspended in 500 lPBS, and the lymphocyte group was determined from the lateral scattering angle. CD4+T cells were used to draw the door, and the percentage of CD4+CD25+Foxp3+Tregs in CD4+T cell group was calculated.
3, double antibody sandwich ELISA was used to detect IFN- gamma, TNF- alpha, IL-6, IL-17, IL-10 and Griess to detect NO2- content in splenocytes culture.
(1) the spleen cells of sample preparation were infected before and after infection in 3D, 5D, 8D of conventional asepsis spleen of mice with 0.17mol/L NH4Cl erythrocyte lysis. With 10%FCS RPMI1640 to adjust the spleen cells at the concentration of 1 * 107/ml in 24 well plates, each hole by adding 500 l of spleen cells suspension at 37 DEG C, under the condition of 5%CO2 culture 48h. note that NA/LE hamster anti-mouse CD3e and purification of natural spleen cells (145-2C11, BD Pharmingen, 0.56 l/well) and hamster anti-mouse (37.51 CD28 purified NA/LE, BD Pharmingen, 0.112 l/well) at 37 DEG C, 5%CO2 under the condition of co culture of 48h to stimulate IL-17 production then, supernatant was collected and stored at -80, the cytokines and NO detection.
(2) double antibody sandwich ELISA kit were detected in the culture supernatant of spleen cells in IFN- gamma, TNF- alpha, IL-6, IL-10, IL-17 secretion, determination of 450nm OD eliasa. Results the standard kit provided by drawing standard curve analysis using SoftMax Pro 4.3.1Ls software, calculate the cytokine levels (pg/ml).
(3) detection of spleen cells NO2- in culture supernatant of Griess method 100 L supernatant and Griess reaction liquid 100 L reaction at room temperature 10min, using NaNO2 as a standard microplate 550nm OD values.
experimental result
1, infection rate, survival rate and disease assessment
Cerebral malaria C57BL/6 mice were injected by P.bANKA blood stage parasite induced toxicity in C57BL/6 mice, paralysis and coma died in 8-11 days after infection, parasitemia compared to 10-20%., BALB/c and DBA/2 mice resistant parasite growth experience similar but not the occurrence of brain lesions, died of anemia and excessive parasitemia in infected 3-4w (60%). From the clinical symptoms score of cerebral malaria, C57BL/6 mice and DBA/2 mice. The score is higher than that of BALB/c
2, dynamic detection of inflammatory cytokines, anti inflammatory cytokines and NO levels during the splenocytes culture supernatant during the infection of mice.
CM susceptible C57BL/6 rats and BALB/c rats with DBA/2 resistance spleen cell culture supernatants of proinflammatory cytokine IFN- gamma, alpha TNF-, IL-6, IL-17 and NO began to increase after infection, infection after 5 Tianda peak, 8 days after infection (C57BL/6 rats showed a slight decline drop). Three types of mouse spleen cell culture supernatant the anti-inflammatory cytokine IL-10 in Tianda after infection 5 peak decreased significantly at 8 days after infection of C57BL/6 rats the level of IL-10, BALB/c and IL-10 levels in rats DBA/2 resistance decreased slightly. The susceptible rat spleen cell culture supernatants of proinflammatory cytokines and NO levels were significantly higher in resistant rats, while the level of IL-10 was significantly lower than that of resistant rats.
3, the percentage and absolute number of Tregs in spleen CD4+T cells at different time points after infection and susceptibility to mice
CM susceptible Tregs percentage and absolute number of CD4+T cells in the spleen of mice and rats began to increase resistance to infection after infection, after 3 Tianda peak, 5 days to 8 days after infection and decreased gradually. During infection, C57BL/6 rat spleen CD4+T cell percentage of Tregs and absolute significantly lower than that of BALB/c and DBA/2 mice.
4, analysis of Tregs effect after CD25 elimination in vivo
The elimination of CD25 C57BL/6, BALB/c DBA/2 and CD4+T cells in the spleen of mice in the percentage of Tregs was significantly lower than in the control rats. Compared with normal mice infected with Tregs, remove the C57BL/6 rat survival rate increased significantly, 5,6,7 day infection rate decreased significantly after infection, clinical symptoms score of CM is reduced, the elimination of CD25 C57BL/6 rats without cerebral malaria in the symptoms of anemia and excessive parasitemia of.CD25 to eliminate the BALB/c and DBA/2 rats and normal rats died of infection similar infection 3-4 weeks after infection, but temporarily control parasitemia during the early stage of infection, the elimination of CD25 Tregs'in 8 days after the rapid recovery, inhibition of T cell activation, unable to control parasitemia, these rats in excessive parasitemia.CD25 died of severe anemia and 3-4 weeks after infection elimination significantly reversed IFN- gamma, alpha TNF-, IL-6, IL-17, infection process and cytokine IL-10 and NO DBA/2 mice with CD25.CD25 to eliminate the elimination of BALB/c mice The process of production is similar.
5, the correlation analysis of Tregs and IFN- gamma, TNF- alpha, NO and IL-10 with insect disease at different time after infection of rats
There was a negative correlation between the number of Tregs and the production of IFN- gamma, TNF- alpha and NO during the infection of brain malaria susceptible and resistant mice. The production of IL-10 was positively correlated with the level of hyponatremia.
conclusion
1, between the pro-inflammatory response and anti-inflammatory response balance for the establishment of severe malaria control was the most important, Tregs is an important regulator to maintain this balance.
2, Tregs by modification of pro-inflammatory response to regulation of pro-inflammatory response and anti-inflammatory response balance mediated CM to the occurrence and outcome of infection.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【共引文献】