【作者】 陈家军；

【导师】 孙宗全；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 心脏移植是治疗终末期心功能衰竭的有效手段,近年手术成功率有较大提高,但急、慢性排斥反应仍是制约移植近、远期疗效的主要因素。随着天然免疫的深入研究,人们认识到Toll样受体(Toll-Like Receptors,TLRs)及其信号途径是联系天然免疫与获得性免疫的重要桥梁,表达TLRs的天然免疫细胞,如树突状细胞(dendritic cell,DC),在移植排斥反应中具有重要作用。本课题拟通过研究TLRs信号途径的表达,从整体、细胞和分子水平探讨和阐明TLRs在心脏移植排斥反应中的作用机制,丰富移植免疫理论;采用针对MyD88的RNAi技术,体外培育稳定的供体未成熟DC,转输受体诱导免疫耐受,探索提高移植心脏存活率的全新手段。本研究分以下四部分:第一部分TLR信号途径在DC成熟中的作用及机制研究实验一小鼠骨髓源性树突状细胞的体外扩增、鉴定及生物学特性分析目的:建立小鼠骨髓源性树突状细胞的体外扩增方法,并进行生物学特性分析与鉴定。方法:以重组小鼠粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素(IL-4)体外诱导小鼠骨髓细胞分化为树突状细胞,分为对照组及LPS组。倒置显微镜动态观察细胞形态学变化,流式细胞术分析细胞表面分子改变,混合淋巴细胞反应检测其刺激T细胞增殖的能力。结果:经体外诱导培养可获得大量DC,并具有典型树突状形态;两组CD11c的表达无显著差别,LPS组CD80、CD86及MHC-Ⅱ分子的表达明显高于对照组;LPS组DC可显著刺激同种异体混合淋巴细胞增殖。结论:体外诱导培养可获得小鼠骨髓来源DC,具有典型树突状细胞的形态及髓系树突状细胞的生物学特性,可广泛应用于临床及实验研究。实验二缺/复氧刺激对小鼠骨髓树突状细胞成熟状态的影响目的:探讨缺/复氧刺激对小鼠骨髓树突状细胞(DC)成熟状态的影响及可能的作用机制。方法:培养小鼠骨髓源性DC,分为对照组及缺/复氧组,对照组在正常培养条件下培养,缺/复氧组给予缺氧气体培养4h,然后在正常培养条件下继续培养1天。应用流式细胞术检测DC细胞表面CD80、CD86、MHC-Ⅱ分子、CD14及TLR4的变化, ELISA法检测DC分泌肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)和白介素-12(IL-12)的浓度,混合淋巴细胞培养检测T细胞增殖能力,免疫细胞化学及Western blot检测DC核因子-κB(NF-κB)的表达。结果:缺/复氧刺激可促进DC高表达CD80、CD86、MHC-Ⅱ分子、CD14及TLR4,促进Th1型细胞因子释放、NF-κB高表达及核移位,并诱导T细胞增殖。结论:缺/复氧刺激可能通过激活TLRs信号途径而促进DC成熟,早期阻抑DC成熟可能成为防治缺血再灌注损伤及器官移植排斥反应的一种新途径。实验三树突状细胞MyD88是Hsp60信号转导的关键分子目的:探讨髓性分化因子88(MyD88)在介导热休克蛋白60(HSP60)激活的Toll样受体4(TLR4)信号途径中的作用及调控树突状细胞(DC)活性的可能机制。方法:培养小鼠骨髓源性DC,分为对照组、HSP60组及RNA干扰组,对照组在正常条件下培养,HSP60组加入终浓度为10μg/ml的HSP60, RNA干扰组于加入MyD88siRNA 12h后给予10μg/ml HSP60刺激,培养48h。免疫细胞化学检测DC MyD88、核因子-κB(NF-κB)的表达,Western blot检测DC MyD88的变化;流式细胞术检测DC细胞表面CD80、CD86及MHC-Ⅱ分子的变化, ELISA法检测各组DC分泌肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)和白介素-12(IL-12)的浓度,混合淋巴细胞培养检测T细胞增殖能力。结果: HSP60可诱导DC高表达CD80、CD86及MHC-Ⅱ分子,促进Th1型细胞因子释放、MyD88高表达及NF-κB核移位,诱导DC成熟并刺激T细胞增殖,MyD88 siRNA可下调DC MyD88的表达并阻断HSP60的这些效应。结论: HSP60通过MyD88依赖的途径活化DC,MyD88是Hsp60信号转导的关键分子,抑制MyD88表达可能是治疗HSP60相关疾病的一种新方法。第二部分TLR信号途径在心脏移植排斥反应中的作用及机制研究目的:检测HSP60及TLR4信号途径小鼠心脏移植物的表达情况,探讨其在心脏移植排斥反应中的作用以它们之间的关系。方法:建立小鼠颈部心脏移植模型,随机分为2组:对照组(同系移植组),供、受体均为C57BL/ 6小鼠;实验组(同种异品系移植组),供、受体分别为BALB/ C、C57BL/ 6小鼠。分别于移植后第3天、第7天取小鼠心脏及血液标本,病检分析心脏组织形态学改变, ELISA法检测小鼠血液肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)和白介素-12(IL-12)的浓度,免疫组化及western blot检测心脏移植物HSP60、TLR4、MyD88及NF-κB的表达情况。结果:病理学检查结果显示实验组小鼠在术后第3、7天分别发生轻、重度排斥,与血清TNF-α、IFN-γ及IL-12的浓度变化相一致,对照组无明显排斥现象;实验组TNF-α、IFN-γ及IL-12的浓度及心脏移植物HSP60、TLR4、MyD88及NF-κB的表达明显高于对照组。结论:心脏移植后HSP60表达增强,可能通过TLR4以MyD88依赖的方式激活TLR4信号途径,从而促进移植排斥反应的发生,调控HSP60其受体后信号途径可能为抗排斥反应提供新的思路。第三部分抑制TLR信号途径对小鼠骨髓DC生物学活性的影响目的:采用RNA干扰技术抑制小鼠骨髓树突状细胞(DC)髓样分化因子88(MyD88)的表达,并检测其对细胞生物学活性的影响,为DC的临床应用奠定基础。方法:针对MyD88基因,采用化学合成法合成3对MyD88 siRNA(序列1、序列2及序列3),并转染DC2.4细胞。采用半定量RT-PCR及Western blot分别从mRNA和蛋白质水平检测DC MyD88的表达情况,筛选其中一对高效RNA(序列3)转染DC2.4细胞(RNA干扰组),以未转染RNA的DC2.4细胞作为对照组,分别给予1mg/L的脂多糖(LPS)刺激。流式细胞术(FCM)检测DC细胞表面CD80、CD86及MHC-Ⅱ分子的变化, ELISA法检测DC分泌肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)和白介素-12(IL-12)的浓度,免疫细胞化学检测DC核因子-κB(NF-κB)的表达,混合淋巴细胞培养检测T细胞增殖能力,观察RNA干扰对LPS促DC成熟的影响。结果:与空白对照组相比,序列2组及序列3组DC的mRNA和蛋白质表达分别降低90%和85%、92%和88%,差异有统计学意义;脂质体对照组、无义siRNA对照组及序列1组DC的mRNA和蛋白质表达无显著差异。经LPS刺激后,与对照组相比,RNA干扰组CD80、CD86及MHC-Ⅱ分子的表达,TNF-α、IFN-γ及IL-12的浓度及T细胞增殖能力均显著下降,且未见明显NF-κB核转位。结论: RNA干扰技术能显著下调小鼠DCMyD88的表达,并显著抑制LPS促DC成熟的效应,为以DC MyD88为靶向的相关疾病的基因治疗提供了新思路和手段。第四部分MyD88siRNA基因修饰的树突状细胞在诱导同种小鼠心脏移植耐受中的作用目的:探讨MyD88siRNA处理的供者来源的树突状细胞(DC)在诱导免疫耐受中的作用。方法:化学合成MyD88siRNA,以脂质体为载体将MyD88siRNA导入BALB/c小鼠骨髓来源DC,制备MyD88siRNA基因修饰的DC(MyD88siRNA-DC)。将MyD88siRNA-DC输至受者C57BL/6小鼠体内,分别于输注后7天、14天和21天应用免疫双荧光染色法观察供者MyD88siRNA-DC在受者脾脏内的存活情况,混合淋巴细胞反应(MLR)测定受者脾脏T细胞对供者同种抗原的反应性。通过袖套管技术建立小鼠异颈部位心脏移植模型,观察心脏移植物存活时间,病理检查观察排斥反应程度,并用ELISA方法测定受者血清Th1型细胞因子(INF-γ和IL-12)和Th2型细胞因子(IL-4和IL-10)水平的变化。结果:输注后14天可见受者脾脏内较多DC存活, MyD88siRNA-DC能诱导受者脾脏T细胞的抗原特异性低反应性,使移植心脏存活天数延长至(24.50±4.42)d,较PBS对照组的(6.67±1.37)d明显延长(P<0.01),降低排斥反应病理分级(Stanford 1~2级),使受者小鼠血清INF-γ和IL-12水平显著降低(P<0.01),明显升高IL-4和IL-10水平(P<0.01)。结论: MyD88siRNA处理的供者来源的DC能够诱导针对移植供者的特异性免疫耐受,其机制可能与诱导受体Th1/Th2免疫偏移及形成供受者微嵌合状态有关。更多还原

【Abstract】 Heart transplantation which is considered as an effective means for treating terminal heart disease has improved greatly rencently , but the acute or chronic immunologic rejection are still a major disturbance which affect operative result. With the development of studying innate immunity, toll-like receptor(TLR) signal pathway is consider as a bridge linked innate immunity and adaptive immunity.The cells which expressed TLR, for example dendritic cell(DC) play important role in allograft rejection. Our project will study the role of TLR signal pathway in cardiac rejection, and try to decrease the morbidity of allograft rejection by inhibiting TLR signal pathway through using MyD88siRNA gene. This study was divided into 4 parts as follows:Role of Toll-like Receptor Signal Transduction In Cardiac Allograft Rejection in MicePart 1.Experiment 1.Study on generation , identification and cytobiological characteristics of dendritic cells from mouse boneObjectives: To obtain and identify dendritic cells(DCs)from mouse bone marrow in vitro and study its cytobiological characteristics. Methods: Mouse bone marrow cells were induced to form dendritic cells by recombinant mouse-granulocyte and macrophage colony-stimulus factor (GM-CSF) and interleukin-4 (IL-4) in vitro. Dendritic cells were divided into control group and LPS group. The morphological changes were observed with light inverted microscope. CD11c, CD80, CD86 and MHC-Ⅱwere identified with flow cytometry. The biological function was studied with mixed lymphocyte reaction (MLR).Results: The cultured DCs from mouse bone marrow displayed the typical morphological characteristics of DCs. The expression of CD1lc is no deference in two groups, while the expression of CD80,CD86and MHC-Ⅱin LPS group are higher than that of control group.The DCs in LPS group could stimulate allogenic mixed lymphocyte proliferation. Conclusions:The DCs generated have cytobiologlcal characteristics of myeloid lineage of DCs,and it will be widely used in the clinical study of and experiment. Experiment 2.The impact of hypoxia /reoxygenation on maturation of dendritic cells cultured from murine bone marrowObjective: To explore the impact and mechanism of hypoxia /reoxygenation stimulation on maturation of dendritic cells( DCs) cultured from murine bone marrow. Methods: Mouse DCs were generated from bone marrow cells and were divided into control group and hypoxia /reoxygenation group. DCs in control group was cultured at normal condition, and in hypoxia /reoxygenation group was cultured at hypoxic condition for 4 hours followed by cultured at normal condition for 24 hours. Flow cytometry and mixed lymphocyte reaction (MLR) was used to detect the phenotype and functional properties of DCs. ELISA was used to detect the concentration of TNF-α, IFN-γand IL-12 in the supernatant. Immunochemistry and western blot was used to detect the concentration of NF-κB. Results: Hypoxia /reoxygen stimulation increased the CD80, CD86, MHC-Ⅱ, CD14 and TLR4 in the cytomembrane of DCs and TNF-α, IFN-γ, IL-12 concentration in the supernatant. Hypoxia /reoxygen stimulation also promoted the shift of NF-κB to karyon. Conclusions: Hypoxia /reoxygen stimulation perhaps promots the maturation of DCs through toll-like receptors signal pathway. Inhibition maturation of DCs may be a novel way for treatment of ischemia-reperfusion injury and rejection.Experiment 3.Role of Myeloid differentiation factor 88 in HSP60 signal transduction in dendritic cellsObjective: To explore the role and mechanism of myeloid differentiation factor 88(MyD88) in HSP60 signal transduction in dendritic cells. Methods: Mouse DCs were cultured from murine bone marrow cells. The DC marker CD11c was detected by flow cytometry, then DCs were divided into control group, HSP60 groupand RNA interference group. Control group was cultured under normal condition, and HSP60 group was cultured with 10μg/ml of HSP60. RNA interference group was first cultured with MyD88 siRNA for12 hours and then HSP60 was added into the culture mixture. All groups were cultured for 48 hours. Immunochemistry was used to detect the concentration of MyD88 and NF-κB. Western blot was used to detect the concentration of MyD88.Flow cytometry and mixed lymphocyte reaction (MLR) were used to detect the phenotype and functional properties of DCs. ELISA was used to detect the concentration of TNF-α, IFN-γand IL-12 in the supernatant. Results: The expression of CD11c in murine bone marrow DCs was 88.76%. HSP60 stimulation increased the expression of CD80, CD86, MHC-Ⅱin DCs and TNF-α, IFN-γ, IL-12 secretion in the supernatant. HSP60 stimulation also increased the level of MyD88 in the cytoplasm and promoted the shift of NF-κB to karyon and the proliferation of allogeneic T cells. MyD88 siRNA could decrease MyD88 and inhibit these effects induced by HSP60. Conclusions: HSP60 activates DCs through MyD88-dependent pathway. MyD88 is a critical role in HSP60 signal transduction. Inhibition of MyD88 may be a novel way for treating disease correlated with HSP60.Part 2. Expression and significance of HSP60 and toll-like receptor 4 transducting system in mouse cardiac transplantationObjective: To detect the expression of HSP60 and toll-like receptor 4 transducting system in mouse cardiac transplantation , and to investigate their role in mouse cardiac transplantation. Methods: Mouse cervical heart transplantation model was established The animals were divided into control group and experimental group . The heart and blood were chosen for study at day 3 and day 7. Pathological analysis were performed.The levels of cytokines in the blood serum were determined using ELISA. The expression of HSP60, TLR4,MyD88 and NF-κB in cardiac transplantation were determined by immunohistochemistry and western blot. Results: Severe rejection could observed in experimental group whereas no distinct rejection in control group. Thl cytokines (TNF-α、IFN-γ,IL-12)increased significantly in experimental group as compared with that in control group. The expression of HSP60, TLR4,MyD88 , NF-κB was higher in experimental group than that in control group. Conclusions: HSP60 increased significantly after heart transplantation which can activate toll-like receptor 4 transducting system in MyD88-dependent pathway and promote allograft rejection. Regulation of HSP60 signal transduction may be a novel way for treating allograft rejection. Part 3.Inhibitory effects of RNA interference on MyD88 expression and biological activity in murine myeloid dendritic cellsObjective: To synthesize small interference RNA aimed directly at myeloid differentiation factor 88 in murine myeloid dendritic cells(DCs) and observe the inhibitory effects on MyD88 expression and biological activity of DCs by RNA interference, and provide basis for clinical applications of DCs. Methods: Three pairs of MyD88 siRNA were synthesized and transfected into DCs with RNAi-mate. The mRNA and protein expressions of MyD88 were analyzed by semi-quantified RT-PCR and Western blot. Mouse DCs were divided into control group and RNA interference group. One of the highest effective siRNA was transfected into RNA interference group. 12 hours later, LPS of the final concentrations of 10μg/ml was added in two groups and continued to culture for 3 days. Flow cytometry and mixed lymphocyte reaction (MLR) were used to detect the phenotype and functional properties of DCs. ELISA was used to detect the concentration of TNF-α, IFN-γand IL-12 in the supernatant. Immunochemistry was used to detect the concentration of NF-κB. Results: MyD88 mRNA and protein were reduced 90% and 85% by sequence2 siRNA, 92% and 88% by sequence3 siRNA respectively, while no change was found in other groups. LPS stimulation increased the CD80, CD86, MHC-Ⅱin the cytomembrane of DCs and TNF-α, IFN-γ, IL-12 concentration in the supernatant in control group. LPS stimulation also promoted the shift of NF-κB to karyon and the proliferation of allogeneic T cells in control group. MyD88 RNA interference can inhibit these effects. Conclusions: RNA interference can knockdown MyD88 expression in murine myeloid dendritic cells and inhibit maturation of DCs. This may provide a new strategy of gene therapy for relative diseases.Part 4.Pretreatment of donor dendritic cells with MyD88siRNA to induce tolerance in mouse allograft recipientsObjective: To explore the effects of donor dendritic cells treated with MyD88siRNA in tolerance induction in mouse allograft recipients. Methods: MyD88siRNA were synthesized chemically and transfected into DCs derived from BALB/c bone marrow by RNAi-mate. The DCs modified with MyD88siRNA,named MyD88siRNA-DC,were injected into the recipient C57BL/6 mice 7 days before transplantation.The existence of the donor MyD88siRNA-DC in the recipient animal spleens was studied by double- immunofluorescence staining.The responsiveness of the recipient spleen T cell to the donor alloantigen was determined by mixed lymphocyte reaction (MLR). The cervical heterotopic heart transplantation model was established with“cuff”technique and the cardiac allograft survival time was observed. Pathological analysis were performed and the levels of cytokines in the serum were determined using ELISA. Results: The survival rate of the donor-derived MyD88siRNA-DC in the recipient spleens was higher than that of the donor derived Day8-DC. The donor-derived MyD88siRNA-DC induced alloantigen-specific T-cell hypo-responsiveness. The cardiac allograft survival time of the MyD88siRNA-DC treated group was longer than that of the Day8- DC group and PBS treated group (24.50±4.42)days vs(13.67±2.25) days and (6.67±1.3) days (P<0.01). Pathological grade of rejection was significantly lower (P< 0.01).In the MyD88siRNA-DC treated group,the levels of IL-12 and IFN-γin the serum decreased significantly(P< 0.01),but the levels of IL- 4 and IL-10 in the serum increased significantly (P<0.01). Conclusions: The injection of the donor-derived MyD88 siRNA-DC can leads to donor-specific tolerance in transplant recipients.The Polarization of Th2 response and chimerism of recipient may play important roles on immune tolerance to cardiac allografts.更多还原