本文關(guān)鍵詞：自然調(diào)節(jié)性T細(xì)胞通過修飾前炎癥應(yīng)答介導(dǎo)腦型瘧疾的發(fā)生　出處：《中國醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 腦瘧 伯氏瘧原蟲ANKA CD4~+CD25~+Foxp3~+調(diào)節(jié)性T細(xì)胞 前炎癥應(yīng)答

【摘要】： 實驗?zāi)康?瘧疾感染是當(dāng)今威脅人類健康的主要難題之一,每年有一百多萬兒童死于瘧疾。腦型瘧疾(cerebral malaria, CM),簡稱腦瘧,是瘧疾最嚴(yán)重的并發(fā)癥之一,而且是瘧疾感染的主要死因。CM嚴(yán)重性的決定因素尚不完全清楚,但很可能來自宿主和寄生蟲兩方面。最終,寄生蟲和免疫應(yīng)答的相互作用決定宿主感染結(jié)局。因此,關(guān)于CM發(fā)生免疫機制的基礎(chǔ)研究是研制有效抗瘧疫苗和藥物的前提。 伯氏瘧原蟲(Plasmodium berghei ANKA,P.bANKA)鼠瘧與人類疾病有許多共同特征,是現(xiàn)有CM的最佳模型。目前研究認(rèn)為CM是由腦血管內(nèi)感染紅細(xì)胞(parasitized red blood cells,pRBC)粘附和細(xì)胞因子及效應(yīng)細(xì)胞介導(dǎo)的強烈的宿主前炎癥應(yīng)答的結(jié)合效應(yīng)引起的疾病。相關(guān)研究表明活化T細(xì)胞介導(dǎo)的免疫應(yīng)答參與疾病誘導(dǎo),并且CD4+T細(xì)胞和CD8+T細(xì)胞均可促進腦病變發(fā)生。事實上,Thl應(yīng)答在控制蟲血癥方面發(fā)揮關(guān)鍵作用,同時也促進CM發(fā)生。 CD4+CD25+調(diào)節(jié)性T細(xì)胞(regulatory T cells, Tregs)是不同于Thl和Th2的具有免疫調(diào)節(jié)效應(yīng)的T細(xì)胞亞群。Tregs活化是寄生蟲逃避宿主免疫的機制之一前期試驗已證明Tregs活化與約氏瘧原蟲(Plasmodium yoelii 17XL, P.yoelii 17XL)感染鼠的易感性相關(guān),Tregs可通過修飾樹突狀細(xì)胞(Dendritic cells, DCs)應(yīng)答,誘導(dǎo)IL-10產(chǎn)生和CD4+T細(xì)胞凋亡調(diào)控Th1應(yīng)答。為此本研究動態(tài)觀察了正常感染鼠及Tregs消除鼠前炎性細(xì)胞因子,抗炎性細(xì)胞因子表達水平和Tregs增殖變化,以期闡明Tregs在腦瘧發(fā)生中的作用地位及其相關(guān)機制。 實驗方法 1、實驗動物模型構(gòu)建及CM病變評估 6-8w,雌性C57BL/6經(jīng)腹腔感染1×106 P.berghei ANKA寄生的紅細(xì)胞(pRBC)構(gòu)建CM易感模型,感染BALB/c和DBA/2鼠構(gòu)建CM抵抗模型。實驗組(6只/組)和對照組(6只/組)小鼠于感染前1d和感染后1d分別經(jīng)腹腔注射1mganti-CD25mAb (7D4,rat IgM; Bio Express)和等體積phosphate-buffered saline (PBS)構(gòu)建Tregs消除小鼠模型。感染后6天起每日監(jiān)測鼠兩次,評價臨床實驗?zāi)X瘧(Experimental cerebral malaria, ECM)癥狀,用以下癥狀確定ECM得分：皮毛豎起、顫抖、肢體癱瘓、痙攣、昏迷。每出現(xiàn)一個癥狀給予1分,累計得分≥4分者考慮為重型瘧疾。 2、流式細(xì)胞術(shù)檢測脾細(xì)胞懸液中CD4+T細(xì)胞群內(nèi)CD4+CD25+Foxp3+Tregs的百分含量 分別于感染前和感染后第3d、5d、8d常規(guī)無菌取出小鼠脾臟,用0.17mol/LNH4C1裂解紅細(xì)胞,以含10%胎牛血清(Fetal calf serum, FCS)的RPMI1640調(diào)整脾細(xì)胞終濃度為1×107/ml。每份樣品用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)進行三色分析,另設(shè)陰性對照管。在流式細(xì)胞儀專用染色管中加入新鮮制備的1×107/ml脾細(xì)胞懸液0.1ml,并用FcγⅢ/Ⅱ封閉抗體孵育以阻斷熒光抗體的非特異染色,再加入抗CD4-FITC和抗CD25-APC mAb進行表面染色,固定透膜后,用抗Foxp3-PE mAb進行胞漿內(nèi)染色,用含1%FCS的PBS洗滌兩次并懸浮于500μlPBS中,流式細(xì)胞儀(Becton Dickinson, USA)進行檢測。以前向和側(cè)向散射角確定淋巴細(xì)胞群,CD4+T細(xì)胞畫門,分析計算CD4+T細(xì)胞群內(nèi)CD4+CD25+Foxp3+Tregs的百分率。 3、采用雙抗體夾心ELISA法檢測脾細(xì)胞培養(yǎng)上清IFN-γ、TNF-α、IL-6、IL-17、IL-10及Griess方法檢測NO2-含量 (1)脾細(xì)胞樣品制備分別于感染前和感染后第3d，5d，，8d常規(guī)無菌取出小鼠脾臟,用0.17mol/L NH4Cl裂解紅細(xì)胞。以含10%FCS的RPMI1640調(diào)整脾細(xì)胞終濃度為1×107/ml，于24孔培養(yǎng)板中,每孔加入500μl脾細(xì)胞懸液于37℃,5%CO2條件下培養(yǎng)48h。需要注意的是天然脾細(xì)胞與純化NA/LE hamster anti-mouse CD3e (145-2C11, BD Pharmingen,0.56μl/well)和純化NA/LE hamster anti-mouse CD28 (37.51, BD Pharmingen,0.112μl/well)于37℃,5%CO2條件下共同培養(yǎng)48h以刺激IL-17產(chǎn)生,隨后收集培養(yǎng)上清,-80℃保存,待細(xì)胞因子及NO檢測。 (2)雙抗體夾心ELISA試劑盒分別檢測脾細(xì)胞培養(yǎng)上清中IFN-γ、TNF-α、IL-6、IL-10、IL-17的分泌水平,酶標(biāo)儀測定450nm處OD值。結(jié)果以試劑盒提供的標(biāo)準(zhǔn)品繪制標(biāo)準(zhǔn)曲線,應(yīng)用SoftMax Pro 4.3.1Ls軟件分析,計算細(xì)胞因子含量(pg/ml)。 (3) Griess方法檢測脾細(xì)胞培養(yǎng)上清中NO2-含量100μl上清和Griess反應(yīng)液100μl室溫反應(yīng)10min,用NaNO2作為標(biāo)準(zhǔn)品,酶標(biāo)儀檢測550nm處OD值。 實驗結(jié)果 1、感染率、幸存率與疾病評估 腦瘧是通過給C57BL/6小鼠注射毒性P.bANKA血液階段寄生蟲誘導(dǎo)的,C57BL/6小鼠于感染后8-11天死于麻痹和昏迷,蟲血癥為10-20%。相比而言,抵抗型BALB/c和DBA/2小鼠經(jīng)歷類似的寄生蟲生長但不發(fā)生腦病變,于感染后3-4w死于貧血和過度蟲血癥(60%)。從腦瘧癥狀的臨床得分來看,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間脾細(xì)胞培養(yǎng)上清前炎性細(xì)胞因子、抗炎性細(xì)胞因子和NO水平的動態(tài)檢測 CM易感C57BL/6鼠和抵抗BALB/c與DBA/2鼠脾細(xì)胞培養(yǎng)上清前炎性細(xì)胞因子IFN-γ、TNF-α、IL-6、IL-17及NO于感染后開始升高,感染后5天達峰值,感染后8天下降(C57BL/6鼠呈現(xiàn)輕微下降)。三種鼠脾細(xì)胞培養(yǎng)上清抗炎性細(xì)胞因子IL-10也于感染后5天達峰值,在感染后8天C57BL/6鼠IL-10水平顯著下降,抵抗BALB/c與DBA/2鼠IL-10水平輕微下降。易感鼠脾細(xì)胞培養(yǎng)上清前炎性細(xì)胞因子和NO水平顯著高于抵抗鼠,而IL-10水平顯著低于抵抗鼠。 3、易感和抵抗小鼠感染后不同時間點脾CD4+T細(xì)胞中Tregs的百分比和絕對數(shù) CM易感鼠和抵抗鼠脾臟CD4+T細(xì)胞中Tregs百分比和絕對數(shù)于感染后開始升高,感染后3天達峰值,感染后5天到8天逐漸下降。并且在感染期間,C57BL/6鼠脾臟CD4+T細(xì)胞中Tregs百分比和絕對數(shù)顯著低于BALB/c和DBA/2小鼠。 4、體內(nèi)CD25消除后Tregs效應(yīng)分析 CD25消除的C57BL/6、BALB/c和DBA/2小鼠脾臟CD4+T細(xì)胞中Tregs百分?jǐn)?shù)顯著低于相應(yīng)對照鼠。與正常感染鼠相比,Tregs消除的C57BL/6鼠幸存率明顯增加,感染后5、6、7天感染率顯著下降,CM癥狀的臨床得分也相應(yīng)減少,CD25消除的C57BL/6鼠不發(fā)生腦瘧癥狀,于感染后3-4周死于貧血和過度蟲血癥。CD25消除的BALB/c和DBA/2鼠與正常感染鼠感染過程相似,但在感染初期蟲血癥得到暫時控制,CD25消除后8天Tregs'快速恢復(fù),抑制T細(xì)胞活化,不能控制蟲血癥,這些鼠于感染后3-4周死于過度蟲血癥和嚴(yán)重貧血。CD25消除明顯逆轉(zhuǎn)IFN-γ、TNF-α、IL-6、IL-17、IL-10及NO產(chǎn)生。CD25消除的DBA/2小鼠與CD25消除的BALB/c小鼠的感染進程和細(xì)胞因子產(chǎn)生過程相似。 5、易感和抵抗鼠感染后不同時間Tregs與IFN-γ、TNF-α、NO及IL-10與蟲血癥的相關(guān)性分析 腦瘧易感鼠和抵抗鼠感染期間Tregs數(shù)量與IFN-γ、TNF-α、NO產(chǎn)生呈負(fù)相關(guān),IL-10產(chǎn)生與蟲體血癥水平正相關(guān)。 結(jié)論 1、前炎癥應(yīng)答和抗炎癥應(yīng)答之間平衡的設(shè)立對控制重型瘧疾發(fā)生至關(guān)重要,Tregs是維持此平衡的重要調(diào)節(jié)因子。 2、Tregs通過修飾前炎癥應(yīng)答從而調(diào)控前炎癥應(yīng)答與抗炎癥應(yīng)答之間的平衡以介導(dǎo)CM的發(fā)生和感染結(jié)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.

【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

【共引文獻】

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