本文選題：Toll樣受體4 + 脂多糖　； 參考：《浙江大學(xué)》2011年博士論文

【摘要】：造血干細(xì)胞移植(hematopoietic stem cell transplantation,HSCT)是當(dāng)今治療惡性血液腫瘤的重要手段,移植物抗宿主病(graft-versus-host disease,GVHD)等移植并發(fā)癥嚴(yán)重影響移植效果和臨床的廣泛應(yīng)用。本項(xiàng)目從臨床和動物實(shí)驗(yàn)?zāi)Ｐ腿胧?對造血干細(xì)胞移植供受者TLR4(Toll-like receptors, TLRs)基因單核苷酸多態(tài)性(Single Nucleotide Polymorphisms,SNP)與移植術(shù)后GVHD發(fā)病率和嚴(yán)重程度以及術(shù)后感染的關(guān)系進(jìn)行了研究。同時(shí)也建立了TLR4基因缺陷的小鼠骨髓移植模型,以期研究TLR4分子表達(dá)對GVHD發(fā)病率和移植生存期的影響,觀察TLR4基因缺陷對DC(dendritic cells,DCs)成熟、抗原遞呈功能和誘導(dǎo)T細(xì)胞異基因免疫反應(yīng)能力的影響,闡明TLR4在GVHD中的分子機(jī)制。本研究將為惡性血液腫瘤移植術(shù)后GVHD的預(yù)后因素和防治的研究提供新的思路,為造血干細(xì)胞移植治療惡性血液病提供新的理論依據(jù),具有重要的研究意義。 關(guān)于TLR4對GVHD的影響,國際上有少量報(bào)道涉及,而且結(jié)論尚有爭議。有學(xué)者認(rèn)為TLR4可能減少GVHD的發(fā)病風(fēng)險(xiǎn),而另外學(xué)者認(rèn)為TLR4會增加或不影響GVHD的發(fā)生。為明確TLR4在異基因移植后GVHD發(fā)生中扮演的角色,我們采用PCR擴(kuò)增直接測序法和聚合酶鏈反應(yīng)-限制性片段多態(tài)性分析技術(shù)(PolymeraseChain Reaction-Restriction Fragment Length Polymorphism,PCR-RFLP)去對208例在我移植中心行異基因造血干細(xì)胞移植術(shù)的病人及相配對的親緣或非親緣供者進(jìn)行TLR4基因Asp299Gly和Thr399Ile兩個(gè)單核苷酸多態(tài)性變異位點(diǎn)的基因型測序。Ncol內(nèi)切酶分析Asp299Gly突變,Hinfl內(nèi)切酶分析Thr339Ile突變,我們研究發(fā)現(xiàn)：在所有被檢測的造血干細(xì)胞移植供受者人群樣本中均沒有檢測到TLR4的Asp299Gly和Thr399Ile基因多態(tài)性存在。我們得出結(jié)論：TLR4基因的多態(tài)性在不同的地區(qū)和人種中分布是不同的,在非洲、歐洲人種中分布相對較高,最高突變報(bào)道出現(xiàn)在伊朗西部人群中,而在其他亞洲人種中分布罕見。我們的實(shí)驗(yàn)證明了在中國尤其是浙江及周邊地區(qū)移植人群中TLR4基因Asp299Gly和Thr399Ile的基因多態(tài)性非常罕見。由于這個(gè)結(jié)果使我們無法用統(tǒng)計(jì)學(xué)方法闡明TLR4基因和GVHD之間的聯(lián)系,因此,我們試圖通過TLR4基因敲除小鼠GVHD模型來證實(shí)這個(gè)假設(shè)：供者或受者的TLR4基因突變可能影響到GVHD的發(fā)生。 我們通過小鼠移植模型探討了TLR4基因在骨髓移植后GVHD的發(fā)生中扮演的角色。GVHD的發(fā)生有3個(gè)因素,其中關(guān)鍵因素是供者淋巴細(xì)胞識別供者或受者的抗原遞呈細(xì)胞(antigen -presenting cell, APC)而活化,轉(zhuǎn)移到靶組織器官對受者的組織器官發(fā)生攻擊,引起組織破壞。樹突狀細(xì)胞作為一類具有最強(qiáng)抗原提呈功能的細(xì)胞群體,在識別和遞呈抗原啟動免疫應(yīng)答、誘導(dǎo)移植排斥中起重要的作用。人們一直認(rèn)為,DC是抗原提呈能力最強(qiáng)的APC,是唯一能夠激活初始型T細(xì)胞的APC,具有激活移植排斥反應(yīng)的作用。 我們利用小鼠同種異基因骨髓移植模型觀察TLR4基因缺陷小鼠DC誘導(dǎo)同種移植免疫耐受效果,并分析了相關(guān)的免疫機(jī)制。通過研究TLR4基因敲除小鼠DC在體內(nèi)外對供者T細(xì)胞成熟、分化和功能的影響以及致耐受作用的不同來了解TLR4在GVHD中所扮演的角色。 BALB/c、C57BL/6小鼠是純系的同種小鼠,這兩種小鼠的主要和次要組織相容性抗原(MHC-Ⅰ、Ⅱ)均不相同,可進(jìn)行同種間移植排斥反應(yīng)的免疫學(xué)研究。TLR4-/-是TLR4基因敲除小鼠其背景與C57BL/6小鼠(TLR4+/+)相同。研究表明,異基因骨髓移植后供者和受者來源的APC均可出現(xiàn)在受者的二級淋巴組織中,供者的T細(xì)胞受體(T cell receptor, TCR)可以識別受者APC(直接遞呈)或供者APC(間接遞呈)呈遞的異基因抗原。我們將實(shí)驗(yàn)設(shè)計(jì)為TLR4+/+和TLR4-/-小鼠分別做供者或受者與BALB/c小鼠之間相互進(jìn)行骨髓移植,了解TLR4-/(?)與TLR4+/+的DC在參與直接遞呈和間接遞呈識別過程中,兩者相比受鼠發(fā)生GVHD的不同。通過觀察我們發(fā)現(xiàn)TLR4-/-小鼠無論作供鼠還是受鼠其移植后的嵌合體與TLR4+/+組相比發(fā)生GVHD程度均較輕、體重下降較慢、臨床GVHD評分較低、肝臟及小腸等器官、組織的受損程度較小,均預(yù)示TLR4-/-小鼠可以誘導(dǎo)機(jī)體產(chǎn)生針對特異性抗原的耐受,導(dǎo)致機(jī)體同種異體器官移植的免疫耐受。此外我們還觀察到TLR4-/-小鼠做供鼠時(shí)移植后受鼠BALB/c發(fā)生GVHD時(shí)間(中位時(shí)間16.1天)較TLR4-/-小鼠做受鼠時(shí)其嵌合體發(fā)生典型GVHD(中位時(shí)間12.3天)的時(shí)間也要退后一些,解剖發(fā)現(xiàn)受鼠肝臟、小腸表面出血點(diǎn)程度更輕；我們認(rèn)為這可能是移植后受體淋巴組織中供體或受體來源的DC所占比例不同所造成的,當(dāng)TLR4-/-作為供鼠時(shí),其提供的DC在移植后2周為嵌合小鼠淋巴組織中主要DC,因其遞呈異基因抗原使T細(xì)胞激活的作用較弱,故誘導(dǎo)發(fā)生GVHD的程度要輕。 在進(jìn)一步的體內(nèi)外實(shí)驗(yàn)中我們比較了TLR4-/-小鼠DC的抗原提呈能力、與同種異基因T細(xì)胞的混合淋巴細(xì)胞反應(yīng)(mixed lymphocyte reaction,MLR)、抑制T輔助細(xì)胞1型(T help cell type 1,Thl)亞群的分化以及體外誘導(dǎo)同種抗原特異性T細(xì)胞低反應(yīng)性等來解釋其發(fā)生移植免疫耐受的免疫機(jī)制。近來的研究認(rèn)為在T細(xì)胞反應(yīng)的早期,當(dāng)童貞T細(xì)胞識別APCs提呈的抗原時(shí),APCs是否能夠表達(dá)足夠的CD86、CD80、CD40等共刺激分子決定了T細(xì)胞是否被完全活化,產(chǎn)生免疫應(yīng)答,或是未被活化導(dǎo)致凋亡或無能。體外實(shí)驗(yàn)我們發(fā)現(xiàn)TLR4-/-可以保護(hù)非成熟型DC不被外源性脂多糖(lipopolysaccharide,LPS)刺激所激活。我們的FACS數(shù)據(jù)表明,給予外源性LPS(1μg/ml)刺激24h, TLR4+/+小鼠Day-5-DC表達(dá)高水平CD80、CD86、CD40和MHC-Ⅱ類分子,顯示出成熟型DC的表型特征,白介素12(Interleukin 12,IL-12)分泌水平明顯升高；而TLR4-/-小鼠Day-5-DC則維持在非成熟型狀態(tài),DC的表型和IL-12分泌在LPS刺激前后幾乎無改變,對T細(xì)胞為低刺激活性。 IL-12是由DC分泌的重要的免疫調(diào)節(jié)因子,可以促進(jìn)干擾素-γ(Interferongamma, IFN-γ)的分泌,引起CD4+T細(xì)胞增殖并向Th1細(xì)胞分化,IL-12的缺乏則T細(xì)胞增殖受抑。TLR4-/-小鼠在LPS刺激后DC的非成熟狀態(tài)明顯抑制了其抗原提呈能力,減弱DC與同種異基因T細(xì)胞的混合淋巴細(xì)胞反應(yīng),引起T細(xì)胞增殖的作用較弱,從而減輕GVHD的發(fā)生。在體內(nèi)實(shí)驗(yàn)我們對骨髓移植后21天小鼠進(jìn)行研究,供鼠是TLR4-/-組小鼠脾細(xì)胞來源的DC其表面共刺激分子CD80、CD86的表達(dá)與供鼠TLR4+/+組相比也明顯降低。TLR4基因的缺失可使DC維持非成熟型狀態(tài),具備對T細(xì)胞的低刺激活性。 一般認(rèn)為CD4+T細(xì)胞Th1亞群向Th2亞群漂移,可以改變移植物局部的免疫反應(yīng),抑制細(xì)胞介導(dǎo)的排斥反應(yīng)。IL-2和IFN-γ是Th1亞群的特征細(xì)胞因子,而IL-10和IL-4是Th2亞群來源的細(xì)胞因子；IL-17為第三個(gè)T細(xì)胞亞群,在預(yù)防胞外病原體中扮演重要角色,其缺乏可導(dǎo)致炎癥的進(jìn)展和嚴(yán)重的自身免疫疾病。 為了探討TLR4-/-小鼠的DC與異基因CD4+T細(xì)胞在混合淋巴細(xì)胞反應(yīng)中對T細(xì)胞分化的影響,我們測定并分析了同種MLR反應(yīng)體外培養(yǎng)體系上清中Th1和Th2來源的細(xì)胞因子,發(fā)現(xiàn)TLR4-/-小鼠來源非成熟型DC與新鮮分離BALB/c小鼠的CD4+T細(xì)胞作MLR后,代表Th1亞群的細(xì)胞因子IFN-γ、IL-2水平明顯降低(p0.01),有意思的是代表Th2亞群的細(xì)胞因子IL-10和IL-4水平也表現(xiàn)為下降(p0.05)。結(jié)果說明TLR4-/-小鼠DC可抑制Th1亞群的增生反應(yīng),但并未誘導(dǎo)Th1亞群向Th2亞群偏移。此外TLR4+/+小鼠MLR上清中IL-17的水平與TLR4-/-組相比顯著升高,說明IL-17對GVHD的進(jìn)展起促進(jìn)作用。我們在MLR實(shí)驗(yàn)中通過流式和羧基熒光素二醋酸鹽琥珀酰亞胺酯(carbox fluorescenceindiacetate succinimidyl ester, CFSE)標(biāo)記檢測發(fā)現(xiàn)TLR4-/- DC對異基因T細(xì)胞的活化作用減弱,與TLR4+/+組相比T細(xì)胞增殖明顯減少；本結(jié)果說明TLR4基因的缺失使DC在體外同種MLR反應(yīng)中能夠維持處于非成熟型狀態(tài),不能充分活化T細(xì)胞,從而誘導(dǎo)T細(xì)胞對同種抗原的低反應(yīng)性。 血清中Th1亞群分泌的細(xì)胞因子與嚴(yán)重的GVHD和死亡率相關(guān)。為了解小鼠移植后體內(nèi)來自Th1亞群和Th2亞群細(xì)胞因子水平的變化；推測TLR4-/- DC在體內(nèi)對T細(xì)胞分化的影響；我們收集了移植后7天嵌合體小鼠的血清,供鼠為TLR4-/-的移植后受鼠血清中代表Th1亞群的IL-2水平明顯較低；而令人驚訝的是其血清中IFN-γ的水平正好相反,移植后第7天受鼠血清中IFN-γ水平與TLR4+/+小鼠組的比較明顯升高,考慮可能是除T細(xì)胞以外的其他細(xì)胞如自然殺傷細(xì)胞(Natural killer cells, NK cells)、APC分泌的較多量IFN-γ所至。這結(jié)果也與其他一些學(xué)者觀點(diǎn)符合：IFN-γ對GVHD起到保護(hù)作用,IFN-γ對活化T細(xì)胞起負(fù)調(diào)控作用,抑制細(xì)胞分化,促進(jìn)細(xì)胞死亡,保護(hù)受體器官不受損壞。研究發(fā)現(xiàn)Th2相關(guān)的細(xì)胞因子可以下調(diào)細(xì)胞介導(dǎo)的免疫反應(yīng),拮抗Th1亞群的細(xì)胞因子效應(yīng),從而減輕GVHD的發(fā)生。我們檢測發(fā)現(xiàn)代表Th2亞群的細(xì)胞因子IL-10水平在供鼠為TLR4-/-的嵌合小鼠血清中表現(xiàn)為明顯升高,提示可以抑制小鼠移植后GVHD的發(fā)生。此外移植后第7天最大量成熟的DC遷移到淋巴組織,參與到遞呈抗原,活化T細(xì)胞的過程中,這一定程度解釋了實(shí)驗(yàn)中發(fā)現(xiàn)移植后嵌合體小鼠血清中細(xì)胞因子檢測為何在第7天水平最高這一現(xiàn)象。 影響GVHD的因素很多,在體內(nèi)這些因素相互關(guān)聯(lián)形成復(fù)雜的網(wǎng)絡(luò)最終影響到GVHD的發(fā)生和嚴(yán)重程度,LPS/TLR4的信號傳導(dǎo)通路也是其中影響因素之一。我們從這個(gè)方面入手了解天然免疫分子TLR4在GVHD中所起的作用,最后得出結(jié)論TLR4基因在T細(xì)胞對APCs的刺激產(chǎn)生明顯的活化和增殖反應(yīng)中起著重要的橋梁作用,是同種反應(yīng)性T淋巴細(xì)胞啟動介導(dǎo)同種免疫應(yīng)答的關(guān)鍵因素；TLR4基因的缺失可誘導(dǎo)機(jī)體產(chǎn)生針對特異性抗原的耐受,導(dǎo)致機(jī)體同種異體器官移植的免疫耐受從而顯著減少受體GVHD的發(fā)生。針對TLR4基因研發(fā)新的靶向治療藥物也許可以為今后預(yù)防和減輕異基因造血干細(xì)胞移植后GVHD的發(fā)生提供了一個(gè)新的治療思路。
[Abstract]:Hematopoietic stem cell transplantation (HSCT) is an important means for the treatment of malignant hematological tumors. Graft versus host disease (graft-versus-host disease, GVHD) and other transplantation complications seriously affect the effect of transplantation and extensive clinical application. This project starts with the clinical and animal model of hematopoiesis. The single nucleotide polymorphisms of TLR4 (Toll-like receptors, TLRs) gene (Single Nucleotide Polymorphisms, SNP) and the relationship between the morbidity and severity of GVHD and the postoperative infection after transplantation were studied. The mouse bone marrow transplantation model of the TLR4 gene defect was also established in order to study the TLR4 molecule expression for GVHD hair. The influence of the rate of disease and the survival time of the transplant, to observe the effect of TLR4 gene defect on the maturation of DC (dendritic cells, DCs), the antigen presenting function and the ability to induce the allogeneic immune response of T cells, and to elucidate the molecular mechanism of TLR4 in GVHD. This study will provide a new idea for the study of the prognostic factors and the prevention and treatment of GVHD after malignant hematological tumor transplantation. It provides a new theoretical basis for hematopoietic stem cell transplantation in the treatment of hematological malignancies, and has important research significance.
There are a few international reports about the impact of TLR4 on GVHD, and there are controversial conclusions. Some scholars believe that TLR4 may reduce the risk of GVHD, while other scholars believe that TLR4 will increase or do not affect the occurrence of GVHD. In order to clarify the role of TLR4 in the occurrence of GVHD after allogeneic transplantation, we use PCR direct sequencing and aggregation. PolymeraseChain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) was used to perform the TLR4 gene Asp299Gly and Thr399Ile two single nucleotide sequences in 208 patients with allogeneic hematopoietic stem cell transplantation at the center of my transplant and the matched or unrelated donors in my transplant center. Asp299Gly mutation and Hinfl endonuclease analysis of Thr339Ile mutations were analyzed by genotype sequencing.Ncol endonucleases of polymorphic polymorphisms. We found that there was no Asp299Gly and Thr399Ile polymorphism in TLR4 in all samples of the donor recipients who were detected. We concluded: TLR4 gene The polymorphism distribution is different in different regions and ethnicity, in Africa, the European ethnicity is relatively high, the highest mutation report appears in the western Iran population, and is rare in other Asian people. Our experiment proved that the TLR4 gene Asp299Gly and Thr39 in the transplant population in China, especially in Zhejiang and the surrounding areas. The genetic polymorphism of 9Ile is very rare. Because this result makes it impossible to statistically clarify the connection between TLR4 and GVHD, we attempt to confirm this hypothesis by the GVHD model of the TLR4 knockout mice. The mutation of the donor or recipient's TLR4 gene may sound to the occurrence of GVHD.
We explored the role of the TLR4 gene in the occurrence of GVHD after bone marrow transplantation by 3 factors, the key factor is the activation of the donor lymphocyte recognition donor or recipient's antigen presenting cell (antigen -presenting cell, APC) and transferred to the target tissue and organs to the tissues and organs of the recipient. As a class of cell populations with the strongest antigen presentation function, dendritic cells play an important role in identifying and presenting antigens to initiate immune responses and induce transplant rejection. It has been believed that DC is the most powerful APC for antigen presentation, and is the only APC capable of activating the initial T cells. The effect of graft rejection.
We used the mouse allogeneic allogeneic bone marrow transplantation model to observe the effect of DC induced allograft tolerance in TLR4 gene deficient mice, and analyzed the related immune mechanisms. By studying the effect of DC on the maturation, differentiation and function of donor T cells in vivo and outside the donor TLR4 gene knockout mice and the difference in tolerance, the TLR4 in GVHD was understood. The role played in it.
BALB/c, C57BL/6 mice are homozygous homologous mice. The main and secondary histocompatibility antigens (MHC- I, II) of these two mice are all different. The immunological study of the allograft rejection reaction is the same as the C57BL/6 mouse (TLR4+/+) in the TLR4 gene knockout mice. The study showed that the donor and the donor after allogeneic bone marrow transplantation were the same. The recipient's APC may appear in the recipient's two stage lymphoid tissue, and the donor's T cell receptor (T cell receptor, TCR) can identify the recipient APC (direct presentation) or the donor APC (indirect presentation) presentation of the allogeneic antigen. We designed TLR4+/+ and TLR4-/- mice to act as donors or recipients and BALB/c mice, respectively. Bone marrow transplantation (BMT) was used to understand the difference between the TLR4-/ (?) and the TLR4+/+ DC in the direct presentation and indirect presentation, compared with the GVHD in the rats. Through observation, we found that the chimerism of the TLR4-/- mice, no matter the donor or the recipient, was lighter than the TLR4+/+ group, and the loss of weight was slower, and the clinical GVHD evaluation was less than that of the TLR4+/+ group. The liver and small intestine and other organs were less damaged, which indicated that the TLR4-/- mice could induce the tolerance of the body to specific antigen, and lead to the immune tolerance of the allogenic organ transplantation. In addition, we also observed that the time of GVHD (16.1 days in the median time) of the rat BALB/c after transplantation was also observed in TLR4-/- mice. The time of the typical GVHD (12.3 days of the median time) of the chimeras at the time of the TLR4-/- mice should also be reduced. The anatomy found that the liver of the rat and the bleeding point on the surface of the small intestine are lighter; we think this may be the result of the different proportion of DC in the donor or receptor source in the recipient lymphoid tissue, when TLR4-/- is used as the donor. In the 2 week after transplantation, the DC is the main DC in the chimeric lymphoid tissue, and the activation of T cells is weak because of its presentation of the allogenic antigen, so the degree of induction of GVHD is light.
In the further experiments, we compared the antigen presenting ability of DC in TLR4-/- mice, the mixed lymphocyte reaction (mixed lymphocyte reaction, MLR) with the allogeneic T cells, the inhibition of the differentiation of the T auxiliary cell 1 (T help cell type 1) subgroup and the induction of the specific antigen specific cells in vitro. The recent study suggests that when the virginal T cells identify the antigen presented by APCs at the early stage of the T cell response, whether APCs can express enough CD86, CD80, CD40 and other co stimulators determines whether T cells are fully activated, produce an immune response, or are not activated to lead to apoptosis or inactivation. In vitro experiments, we found that TLR4-/- can protect immature DC from exogenous LPS (lipopolysaccharide, LPS) stimulation. Our FACS data show that exogenous LPS (1 mu g/ml) stimulates 24h, TLR4+/+ mice Day-5-DC expresses high level CD80, CD86, CD86, and class II molecules, showing the phenotypic characteristics of mature type. The secretion level of prime 12 (Interleukin 12, IL-12) increased significantly, while Day-5-DC in TLR4-/- mice was maintained in the non mature state. The phenotype of DC and the secretion of IL-12 were almost unchanged before and after LPS stimulation, and the activity of T cells was low.
IL-12 is an important immunomodulatory factor secreted by DC, which can promote the secretion of interferon - gamma (Interferongamma, IFN- gamma), cause CD4+T cells to proliferate and differentiate into Th1 cells. The proliferation of IL-12 in the proliferation of T cells inhibits the antigen presenting ability of the.TLR4-/- mice in DC after LPS stimulation, and reduces the DC and isobasis. Because of the mixed lymphocyte reaction of T cells, the effect of T cell proliferation is weak, thus reducing the occurrence of GVHD. In vivo experiment, we studied the mice 21 days after bone marrow transplantation, and the rat was the DC surface CO stimulator of DC in the TLR4-/- group, and the expression of CD86 was significantly lower than that of the donor TLR4+/+ group. The absence of DC can maintain a non mature state and have a low activation of T cells.
It is generally believed that the Th1 subgroup of CD4+T cells drifts to the Th2 subgroup, which can change the local immune response to the graft, inhibit the cell mediated rejection of.IL-2 and IFN- gamma as the characteristic cytokine of the Th1 subgroup, while IL-10 and IL-4 are the cytokines of the Th2 subgroup, and IL-17 is the third T subsets that play an important role in the prevention of extracellular pathogens. Lack of color can lead to inflammation and severe autoimmune diseases.
In order to investigate the effect of DC and CD4+T cells in TLR4-/- mice on the differentiation of T cells in mixed lymphocyte reaction, we measured and analyzed the cytokines of Th1 and Th2 in the supernatant of the culture system of the same MLR reaction in the culture system, and found that the non mature DC from TLR4-/- mice and CD4+T cells of the newly separated BALB/c mice were substituted for MLR. The cytokine IFN- gamma and IL-2 level of the subgroup of table Th1 were significantly decreased (P0.01). It was interesting that the level of cytokine IL-10 and IL-4 representing Th2 subgroup was also decreased (P0.05). The results showed that DC in the TLR4-/- mice could inhibit the proliferation reaction of Th1 subgroup, but did not induce the Th1 subgroup to shift to the subgroup. The level of IL-17 was significantly higher than that in the TLR4-/- group, indicating that the progress of GVHD was promoted. In the MLR experiment, we found that the activation of TLR4-/- DC on the allogeneic T cells was weakened by the flow and carboxyl fluorescein two acetate succinimide (carbox fluorescenceindiacetate succinimidyl ester, CFSE) markers. The proliferation of T cells was significantly reduced, and the results showed that the deletion of the TLR4 gene could keep the DC in the non mature state in the MLR reaction in vitro, and not fully activate the T cells, thus inducing the low responsiveness of the T cells to the same antigen.
The cytokines secreted by the Th1 subgroup in the serum are associated with severe GVHD and mortality. To understand the changes in the level of Th1 subsets and Th2 subsets in the mice after transplantation, and to speculate on the effect of TLR4-/- DC on the differentiation of T cells in the body; we collected the serum of the chimeric mice 7 days after transplantation, and the mice were transplanted to TLR4-/- after transplantation. The IL-2 level of the Th1 subgroup in the rat serum was significantly lower, but it was surprising that the level of IFN- gamma in the serum was just the opposite. The level of IFN- gamma in the rat serum was significantly higher than that in the TLR4+/+ mice seventh days after the transplantation, considering that other cells except the T cells such as the natural killer cells (Natural killer cells, NK cells). APC secretes more IFN- gamma. This result also conforms to some other scholars' Views: IFN- gamma plays a protective role on GVHD, IFN- gamma plays a negative regulatory role in activating T cells, inhibits cell differentiation, promotes cell death, and protects receptor organs from damage. The study found that the cytokine of Th2 phase can reduce cell mediated immune responses. We should antagonize the cytokine effect of the Th1 subgroup and reduce the occurrence of GVHD. We found that the level of cytokine IL-10 representing the Th2 subgroup was significantly elevated in the mouse sera of the chimeric mice fed as TLR4-/-, suggesting that the occurrence of GVHD in mice after transplantation was inhibited. In addition, the most mature DC migrated to the lymphoid group at seventh days after transplantation. In the process of participating in the delivery of antigen and activating T cells, this explains to some extent the phenomenon that the detection of cytokines in the serum of chimeric mice after transplantation is the highest at the seventh day level.
There are many factors affecting the GVHD, which are related to the formation of complex networks in the body and ultimately affect the occurrence and severity of GVHD. The signal transduction pathway of LPS/TLR4 is also one of the influencing factors. We begin with this aspect to understand the role of the natural immune molecule TLR4 in GVHD, and finally conclude that the TLR4 gene is in T. Cells play an important role in the activation and proliferation of APCs stimulation. It is the key factor for the homologous immune response to the activation of the homologous T lymphocyte, and the deletion of TLR4 gene can induce the specific specificity of the organism.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 扈江偉;金建剛;王莉莎;寧紅梅;俞立權(quán);馮凱;陳虎;;異基因骨髓移植小鼠GVHD模型的建立[J];軍事醫(yī)學(xué)科學(xué)院院刊;2007年02期

2 鄭高鋒;蔡真;黃河;李黎;謝萬灼;施繼敏;何靜松;林茂芳;;血漿置換對異基因造血干細(xì)胞移植術(shù)后急性移植物抗宿主病治療評價(jià)[J];實(shí)用腫瘤雜志;2005年06期

3 黃文燁,蔡真,孫文佶,陸國華;霉酚酸對小鼠樹突狀細(xì)胞體外成熟和免疫功能的影響[J];中國實(shí)驗(yàn)血液學(xué)雜志;2004年02期

4 黃河,蔡真,林茂芳,謝萬灼,梁彬,李黎,何靜松,羅依,鄭偉燕,張潔,葉t錦,胡曉蓉,陳水云,金愛云;非親緣異基因骨髓移植治療兒童白血病[J];中華兒科雜志;2004年11期

5 ;Influence on the immune function of the human peripheral blood mononuclear cells transfected by retrovirus-mediated HSV-tk gene[J];Chinese Medical Journal;2006年12期

6 孟筱堅(jiān);林茂芳;蔡真;;受者特異性產(chǎn)生干擾素γ細(xì)胞與急性移植物抗宿主病的關(guān)系[J];中華血液學(xué)雜志;2006年02期

7 薛興奎;孫文佶;姚航平;胡潔;蔡真;;單純皰疹病毒胸苷激酶基因轉(zhuǎn)染對T細(xì)胞活化和亞群分布的影響[J];中華血液學(xué)雜志;2006年11期

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