小鼠骨髓未成熟树突状细胞体外扩增及鉴定

目的建立体外大量扩增小鼠未成熟树突状细胞(DC)的方法，从形态学、免疫表型和细胞功能试验等方面予以鉴定。方法制备小鼠骨髓细胞，分别用不同剂量重组小鼠粒细胞巨噬细胞集落刺激因子(rmGM—CSF)培养，7d后收集悬浮细胞进行扫描电镜观察和免疫表型鉴定，并行混合淋巴细胞反应，观察其诱导未致敏T淋巴细胞增殖的情况。结果小剂量rmGM—CSF培养获得的DC(GM^low DC)具有DC的典型特征，细胞表面高表达CD11c，低表达CD40、I—A／1-E，不表达B7—1，与大剂量rmGM—CSF培养获得的DC(GM^high DC)相比，其体外刺激未致敏T淋巴细胞增殖的能力较弱。结论本实验中获得的GM^low DC形态上具有DC的典型特征，在细胞表型、细胞功能试验上具有未成熟的特性，说明所建立的培养未成熟DC的方法是可行的；rmGM—CSF的剂量与细胞的成熟程度相关，一般说来，较大剂量的rmGM—CSF诱导生成的细胞以成熟。DC为主，小剂量rmGM—CSF诱导生成的细胞以未成熟DC为主。     

大鼠未成熟树突状细胞体外扩增及功能鉴定

摘要：目的 探讨建立大鼠体外大量扩增未成熟树突状细胞(DC) 的方法, 以及不同剂量粒细胞巨噬细胞集落刺激因子(GM CSF)对大鼠DC分化成熟的影响。方法 分离纯化并扩增大鼠骨髓细胞，用不同剂量GM CSF培养,6 d 和10 d后收集悬浮细胞进行扫描电镜观察和免疫表型鉴定,并行混合淋巴细胞反应,观察其诱导未致敏T 淋巴细胞增殖的情况。结果 小剂量GM CSF 培养获得的DC(GMlowDC) 形态上具有DC 的典型特征,在细胞表型、细胞功能试验上具有未成熟的特性,具有DC 的典型特征,细胞表面高表达CD11c,低表达CD80，CD86及MHC II类分子,与大剂量GM CSF加IL 4的联合组培养获得的DC( GMhighDC) 相比,其体外刺激未致敏T 淋巴细胞的增殖能力较弱.结论 笔者所建立的培养未成熟DC 的方法是可行的；GM CSF的剂量与细胞的成熟程度相关。     

低剂量粒巨噬细胞集落刺激因子培养小鼠骨髓源性未成熟树突状细胞的实验研究

目的建立小鼠骨髓源性未成熟树突状细胞(DC)的培养方法,并初步观察其生物学特性。方法分别用常规剂量和低剂量粒巨噬细胞集落刺激因子(GM-CSF)培养小鼠骨髓源性DC,对其进行形态学观察和细胞表型检测,检测其在体外刺激同种异体T细胞增殖的能力,同时比较不同剂量GM-CSF下培养细胞内IL-10和TGF-βmRNA的表达水平。结果常规剂量GM-CSF培养出的DC为成熟DC和未成熟DC的混合体,经脂多糖(LPS)刺激后迅速分化成熟,表型为CD11c+、CD25±、CD80+、CD86+、MHCⅡhi,在体外可强烈刺激同种异体T细胞分化增殖;而低剂量GM-CSF培养出的DC为较单纯的未成熟DC,LPS不能刺激其分化成熟,表型为CD11c+、CD25-、CD80-、CD86-、MHCⅡlow,不能有效活化刺激同种异体T细胞,其IL-10mRNA表达水平明显高于其他细胞。结论用低剂量GM-CSF可培养出表型和功能均未成熟的小鼠骨髓源性DC,其自身高水平分泌IL-10可能是其成熟障碍的原因。     

小鼠骨髓源性树突状细胞的体外诱导培养及功能分析

目的:建立小鼠骨髓源性树突状细胞(DCs)体外培养和扩增方法,为以DCs为靶点的免疫治疗研究提供基础。方法:从C57BL/6小鼠股骨和胫骨中提取骨髓细胞,以含重组小鼠粒细胞-巨噬细胞集落刺激因子(GM-CSF,400U/ml)和IL-4(200U/ml)的RPMI 1640(含10%FBS)进行诱导培养,于培养第4天加入脂多糖(LPS)继续培养2d。利用倒置相差显微镜和透射电子显微镜观察细胞生长状态和超微结构,流式细胞术鉴定培养第6天时DCs纯度及免疫表型,流式细胞微球捕获芯片技术检测第2、4、6天培养上清中细胞因子IL-6、IL-10、巨噬细胞趋化因子-1(MCP-1)、干扰素-1(IFN-1)、TNF-α、IL-12p70含量。结果:重组小鼠GM-CSF和IL-4诱导培养小鼠骨髓细胞4d,加入脂多糖(LPS)继续培养2d,近90%细胞高表达DCs特征性标志物CD11c,高表达抗原提呈分子MHC-Ⅱ和共刺激分子CD86、CD80,具有典型的树突状形态学特征和分泌炎症性细胞因子IL-6、IL-10、MCP-1、TNF-α和IL-12p70的功能。结论:小鼠骨髓细胞以重组小鼠GM-CSF和IL-4体外诱导培养的DCs具有较高的纯度,保持了体内的形态学、免疫表型及功能特征。     

调节性树突状细胞对大鼠异体肢体移植存活时间的影响

目的探讨输注供体、受体调节性树突状细胞对大鼠同种异体移植肢体存活时间的影响。方法在树突状细胞（Dendritic cells，DC）培养过程中加入重组大鼠白细胞介素10（rrIL-10）和转化生长因子β1（TGF-β1），诱导调节性DC。实验受体为SD大鼠，供体为Wistar大鼠，共分4组，每组7只。A组为对照组；B组为直接识别干预组，输注供体调节性DC至受体大鼠；C组为间接识别干预组，输注负载供体抗原的受体调节性DC；D组为联合干预组，同时输注供体调节性DC及负载供体抗原的受体调节性DC。1周后运用显微外科方法行大鼠同种异体肢体移植，观察移植肢体存活情况，并行受体大鼠免疫状态检测。结果B、C组通过输注调节性DC，移植肢体的存活时间得到延长，D组移植肢体的存活时间进一步延长（P〈0．01），并且D组受体的脾细胞产生了对供体细胞的抗原特异性低反应。结论通过联合输注供体调节性DC和负载供体抗原的受体调节性DC，移植肢体的存活时间明显延长。     

GM-CSF调节树突状细胞对移植免疫应答的影响

目的　观察GM CSF对移植免疫应答的影响 ,探讨树突状细胞 (DC)亚群在其中的作用。方法　BALB/C小鼠每日给予 10 μgrhGM CSF ,皮下注射 ,6h后收集外周血 ,分离DC ,流式细胞技术检测DC1(CD8a CD11c ) /DC2 (CD8a CD11c )的比例变化 ;再给予C5 7BL/ 6的外周血单个核细胞 (PBMC)致敏后 ,检测小鼠Th1和Th2细胞因子的变化 ,进行单向混合淋巴细胞反应(MLR) ;并且行小鼠同种异体心脏移植了解移植免疫应答的变化。结果　rhGM CSF刺激后 ,外周血DC1与DC2的比值显著升高 (17.9± 7.6vs 3 .9± 1.2 ,P <0 .0 1) ,体内Th1细胞因子水平明显升高 ,MLR强度增强 ,同种异体小鼠移植心脏存活时间缩短 [(6.2± 1.2 )dvs (7.7± 1.0 )d(P <0 .0 5 ) ]。结论　GM CSF体内应用可以增加DC1绝对数和其与DC2的比值 ,使机体免疫应答向Th1方向倾斜 ,增强移植免疫排斥反应。     

耐受性树突状细胞延长大鼠移植脾存活时间

目的 观察供者来源的耐受性树突状细胞（DC）在脾移植中的作用，并探讨其作用机理。方法 以Wista大鼠为供者，SD大鼠为受者，建立同种颈部异位脾脏移植模型。（1）分离供者的骨髓细胞，分别采用白细胞介素4（IL-4）和白细胞介素10（IL-10）诱导培养出成熟的DC和耐受性DC，并在光镜下观察两者的细胞形态学差别。采用流式细胞术检测两者对共刺激分子CD86表达的差异，采用混合淋巴细胞反应比较其在体外刺激同种异体T淋巴细胞增殖的反应能力。（2）将受者随机分成4组，每组10只。单纯移植组：受者不经任何预处理仅进行脾移植。IL-10DC组：在移植前7d经受者尾静脉注射2×10^6／ml的经IL-10诱导的DC 1 ml。IL-4 DC组：在移植前7d经受者尾静脉注射2×10^6／ml的经IL-4诱导的DC 1 ml。空白对照组：在移植前7d经受者尾静脉注射无细胞的培养液1ml。观察各组移植术后发生急性排斥反应的时间。结果 （1）经IL-10诱导的骨髓细胞表现为未成熟树突状细胞的形态和特性，细胞体积大，但少见树突状突起，细胞表面低表达CD86分子，不能有效刺激T淋巴细胞的增殖。而经IL-4诱导的骨髓细胞为典型的成熟树突状细胞，细胞胞体大，并有树突状突起，细胞表面高表达共刺激分子CD86，可显著刺激T细胞的增殖。（2）IL-10 DC组发生急性排斥反应的时间较其他3组明显延迟（P〈0．01）；IL-4 DC组发生急性排斥反应的时间较单纯移植组和空白对照组明显提前（P〈0．05）；而单纯移植组与空白对照组间则无明显差异（P〉0．05）。结论 应用供者来源的耐受性树突状细胞能够延缓大鼠移植脾急性排斥反应的发生时间。     

异体肢体移植中树突状细胞与免疫耐受

异体肢体移植手术的成功，使人们更关注免疫耐受的研究。树突状细胞作为耐受原可以从多个方面诱导耐受，因此日益受到人们的重视，本文综述了近年来肢体移植中树突状细胞与免疫耐受的研究进展。     

采用经姜黄素处理的未成熟树突状细胞延长大鼠移植肾存活时间

目的 探讨姜黄素(Cur)处理的树突状细胞(DC)诱导同种T淋巴细胞低反应性的效果以及对大鼠移植肾存活时间的影响.方法 体外培养Wistar大鼠骨髓来源的DC,经Cur处理后,以流式细胞仪检测细胞CD11c、CD80、CD86及主要组织相容性复合物(MHC)Ⅱ类抗原的变化,酶联免疫吸附试验测定DC分泌白细胞介素12(IL-12)的水平,混合淋巴细胞反应(MLR)检测其刺激Lewis大鼠T淋巴细胞增殖的能力,二次MLR测定其诱导的T淋巴细胞抗原特异性低反应性.以Wistar大鼠为供者,Lewis大鼠为受者,进行肾移植.术前第7天,经尾静脉给受者输注用Cur处理的供者DC,分设不处理对照组和未成熟DC对照组(经尾静脉注射供者的未成熟DC),术后观察移植肾存活时间及组织学改变情况,第14天检测受者T淋巴细胞对供者成熟DC的反应性.结果 Cur能明显抑制DC共刺激分子CD11c、CD80、CD86及MHCⅡ类抗原的表达以及IL-12的分泌(P＜0.05).同种T淋巴细胞对经Cur处理过的DC刺激的增殖能力明显减低,且这种低反应性具有抗原特异性.对照组和未成熟DC对照组移植肾的存活时间分别为(8.6±2.1)d和(22.4±7.4)d,实验组为(31.5±6.9)d,实验组移植肾存活时间明显长于对照组和未成熟DC对照组(P＜0.05),且其移植肾组织的损伤程度最轻.实验组受者的T淋巴细胞对供者成熟DC刺激的反应性明显低于对照组(P＜0.05),而对第三方无关抗原的刺激保持较高增殖强度.结论 Cur能抑制DC成熟功能,诱导供者特异性的T淋巴细胞低反应性,移植前输注经Cur处理的未成熟DC能显著延长大鼠移植肾的存活时间.     

膀胱癌患者外周血树突状细胞的体外培养扩增和鉴定

目的研究膀胱癌患者外周血树突状细胞(DC)的体外培养扩增和鉴定。方法应用Ficoll-Hypaque离心获得界面细胞,贴壁培养2h,获得单个核细胞,体外以重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF,50ng/ml)+重组人白细胞介素4(rhlL-4,10ng/ml)+人肿瘤坏死因子-α(hTNF-α,50ng/ml)诱导培养。倒置相差显微镜及扫描电镜下观察DC生长情况；流式细胞仪检测DC表型；MTT法检测DC活化的T细胞对肿瘤细胞BIU87的杀伤率。结果第6天体外培养的DC由贴壁状态变为悬浮毛刺状细胞,第8天为形态不规则的毛刺状,为典型DC形态。流式细胞仪检测表明,成熟DC高水平表达CD_(1a)、CD_(83)、CD_(86)及HLA-DR等。被DC激活的T细胞对膀胱癌细胞株BIU87的杀伤率为(48.8±3.7)%,未经DC激活的T细胞的杀伤率为(25.7±1.5)%,2组比较差异有统计学意义(P＜0.01)。结论膀胱癌患者外周血经rhGM-CSF+rhIL-4+hTNF-α体外诱导培养能诱导出DC。     

Local Granulocyte-Macrophage Colony-Stimulating Factor Improves Incisional Wound Healing in Adriamycin-Treated Rats

Purpose Neoadjuvant treatment is often given for locally advanced malignancies; however, clinical and experimental studies have shown that some chemotherapeutic agents impair wound healing. It has been reported that granulocyte-macrophage colony-stimulating factor (GM-CSF) applied locally improves dermal wound healing. Thus, we investigated the effects of locally injected GM-CSF on abdominal wounds impaired by adriamycin, a widely used chemotherapeutic agent. Methods We divided 120 female Sprague-Dawley rats into five treatment groups of 24 rats. Group 1 received saline 8 mg/kg intravenously (i.v.) + laparotomy 14 days later (control); group 2 received 8 mg/kg i.v. adriamycin + laparotomy 14 days later; group 3 received adriamycin 8 mg/kg i.v. + laparotomy + local GM-CSF 50 μg 14 days later; group 4 received saline 8 mg/kg i.v. + laparotomy + local GM-CSF 50 μg 14 days later; and group 5 received adriamycin 8 mg/kg i.v. + laparotomy + systemic GM-CSF 50 μg 14 days later. Sutures were removed on postoperative day (POD) 7 in all five groups, and the abdominal bursting pressures were measured and recorded. Tissue samples were taken from the incision line for histopathological evaluation and hydroxyproline content measurement. Results The bursting pressure was significantly lower in groups 2 and 5 than in groups 1, 3, and 4. The hydroxyproline content and histopathological findings supported this result. Conclusion The local injection of GM-CSF improved impaired wound healing in adriamycin-treated rats.     

Prolongation of Heart Allograft Survival by Immature Dendritic Cells Generated from Recipient Type Bone Marrow Progenitors

Recent studies suggest that particular dendritic cells (DC) subpopulations may be tolerogenic. To test the capacity of different DC subpopulations to modulate allograft rejection, we generated two distinct populations of rat bone marrow-derived DCs (BMDC) with low doses of GM-CSF and IL-4. The non-adherent population (nBMDC), which are the 'classical' DCs was able to stimulate naive allogeneic T cells and could be induced to completely mature using various stimuli. In contrast, the adherent population (aBMDC), which displayed an immature phenotype, was unable to stimulate T cells and was more resistant to maturation. We found that syngeneic aBMDCs, injected one day before transplantation, induced significant prolongation of heart allograft survival and decreased anti-donor humoral and cellular responses. Similarly, syngeneic aBMDCs inhibited T-cell responses to KLH in the spleen but not in lymph node in a KLH immunization model without graft. This effect was not antigen specific and could be reversed using an inhibitor of inducible nitric oxide synthase. This compartmentalized inhibition could be in part explained by the fact that the majority of syngeneic adherent cells administered intravenously were found in the spleen with some of them reaching the T-cell areas. These data suggest that syngeneic aBMDCs can modulate immune responses.     

Rapamycin-Treated, Alloantigen-Pulsed Host Dendritic Cells Induce Ag-Specific T Cell Regulation and Prolong Graft Survival

Tolerogenic properties of dendritic cells (DC), particularly those in the immature state, and their therapeutic potential are increasingly being recognized. Among several distinct approaches to generate stably immature DC, pharmacologic manipulation stands out as a promising and clinically applicable option. We have shown recently that the immunophilin ligand rapamycin (Rapa) can inhibit DC maturation and their effector functions. Here, we examined the impact of Rapa exposure on subsequent alloantigen (Ag) presentation by myeloid DC via the indirect pathway. Rapa-treated, allogeneic lysate-pulsed host DC (Rapa-DC) were inferior stimulators of syngeneic T cells, compared to lysate-pulsed control DC. Rapa exposure did not block alloAg uptake by DC nor impair their in vivo homing to splenic T cell areas after adoptive transfer. T cells primed by Rapa-treated, alloAg-pulsed DC showed decreased capacity to produce IL-2 and IFNgamma, and were hyporesponsive to subsequent challenge via both the direct and indirect pathways, in an Ag-specific manner. When infused 1 week before transplantation, these Rapa-DC significantly prolonged alloAg-specific heart graft survival. This effect was reversed by systemic IL-2 administration but enhanced by either repeated infusion of the cells or a short post-transplant course of FK506. These therapeutic effects, achieved by targeting both major pathways of allorecognition, provide the basis for a clinically applicable strategy to suppress graft rejection.     

Preferential Depletion of Blood Myeloid Dendritic Cells During Acute Cardiac Allograft Rejection Under Controlled Immunosuppression

Allo-Ag presentation to Ag-specific T-lymphocytes by donor or recipient dendritic cells (DCs) induces acute rejection (AR) after solid organ transplantation. It is postulated that myeloid (mDC) and plasmacytoid (pDC) subsets circulate differentially between bone marrow, heart and lymphoid tissues after cardiac transplantation (HTx). We investigated peripheral blood DC subset distribution, maturation and lymphoid homing properties in relation to endomyocardial biopsy (EMB) rejection grade after clinical HTx. Twenty-one HTx recipients under standard immunosuppression were studied in a 9-month follow-up. mDC and pDC numbers were analyzed by flow cytometry in fresh venous whole blood samples collected during the EMB procedures and before histological diagnosis of AR. Subsets were further characterized for maturation marker CD83 and lymphoid homing chemokine receptor CCR7. Although numbers of both DC subsets remained low for the whole post-HTx period, we observed a negative association of mDCs with rejection grade. Repeated measurements analysis revealed that only mDCs decreased during AR episodes. Rejectors had lower mDC numbers after a 3-month follow-up compared to nonrejectors. Furthermore, patients during AR exhibited low proportions of mDCs positive for CD83 or CCR7. These findings suggest peripheral blood mDC depletion in association with selective lymphoid homing of this subset during AR after clinical HTx.     

Effect of Stem Cell Factor and Granulocyte-Macrophage Colony-Stimulating Factor-Induced Bone Marrow Stem Cell Mobilization on Recovery from Acute Tubular Necrosis in Rats

Background: Acute tubular necrosis (ATN) is the most common reason for acute kidney injury (AKI), and there is still an absence of effective therapies. Objective: To assess the value of bone marrow cell mobilization by stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) therapy in rats with gentamicin-induced ATN. Methods: ATN was induced in male Sprague–Dawley (SD) rats with five daily high-dose intraperitoneal injections of gentamicin. Subcutaneous injections of SCF and GM-CSF were administered simultaneously and these cytokines were observed on days 2, 5, 10, 17, 24, and 31. Peripheral blood and renal tissue CD34+ cell count, mortality rate, blood urea nitrogen (BUN), serum creatinine (SCr), creatinine clearance rate (CCr), and histopathologic lesion scores were determined. Twelve hours after bone marrow ablation (BMA) by lethal X-ray radiation, specific pathogen-free (SPF) ATN rats were given five daily injections of SCF and GM-CSF. BUN, SCr, and histopathologic lesion scores were evaluated on days 2, 5, and 10. Results: Peripheral blood CD34+ cell count increased significantly in ATN rats between 2 and 10 days after SCF and GM-CSF injection. Mortality was reduced from 34.7% in the ATN group to 18.6% in the ATN+CSF. In addition, cytokines administration significantly decreased SCr and BUN. Moreover, cytokines rapidly ameliorated tubular injury. There was no significant effect on ATN rats after BMA. Conclusions: This study demonstrated that SCF and GM-CSF effectively mobilized bone marrow cells in ATN rats, and cytokines administration partially prevented gentamicin-induced ATN. These results suggest that bone marrow stem cell (BMSC) mobilization may be an effective therapy for ATN.     

Primary Alloproliferative TH1 Response Induced by Immature Plasmacytoid Dendritic Cells in Collaboration with Myeloid DCs

The role played by dendritic cell (DC) subsets in the immune response to alloantigens is not well defined. In vitro experiments have extensively shown that freshly isolated myeloid (M)DCs induce a strong T lymphocyte proliferation whereas plasmacytoid (P)DCs do not, unless activated by CD40 ligation. The aim of these studies was to explore whether the interplay among PDCs, MDCs and T cells modulates alloresponse. Freshly isolated MDCs and PDCs were merged in different proportions and used as antigen presenting cells (APCs) in mixed lymphocyte cultures (MLC). As described, isolated PDCs only induced a mild alloresponse, while MDCs were potent inducers of alloproliferation. Unexpectedly, when PDCs were merged with even low numbers of MDCs (down to 100 cells) and used as APCs, a potent Th1 cell proliferation was detected. Survival and maturation of PDCs was increased in these MLC conditions, which could partially explain the magnitude of the T-cell response. Interestingly, the proportion of IFNgamma-producing cells generated in such cultures was higher compared to MDC-stimulated cultures. These data suggest that the interaction between both DC subsets is determinant to generate a potent Th1 response, at least in an allogeneic situation, and may be relevant to the outcome of allogeneic stem cell transplantation.     

胰腺癌患者外周血中树突状细胞的分离、纯化与扩增

目的 探讨从胰腺癌患者外周血中分离、纯化与扩增树突状细胞（DC）的有效方法。方法 分别从健康人（10例，对照组）和胰腺癌患者（12例，实验组）外周血中分离出单核细胞，而后将实验组的单核细胞与GM－CSF及IL－4共同培养，用免疫荧光法和流式细胞仪检测培养前、后的DC数量及DC表面HLA－DR及B7－2的表达水平，并与对照组比较。结果 与对照组相比，实验组DC表面HLA－DR及B7－2表达水平较低（P＜0.01）；经GM－CSF及IL－4联合培养7天后，其单核细胞中的DC数较培养前明显增多（P＜0.01），且DC表面HLA－DR及B7－2表达水平较培养前明显增高（P＜0.01），与对照组相比则无明显差异（P＞0.05）。结论 GM－CSF与IL－4的联合应用能有效地从胰腺癌患者外周血中制备出大量具有功能活性的DC。     

Anti‐Complement Component C5 mAb Synergizes with CTLA4Ig to Inhibit Alloreactive T cells and Prolong Cardiac Allograft Survival in Mice

While activation of serum complement mediates antibody‐initiated vascular allograft injury, increasing evidence indicates that complement also functions as a modulator of alloreactive T cells. We tested whether blockade of complement activation at the C5 convertase step affects T cell‐mediated cardiac allograft rejection in mice. The anti‐C5 mAb BB5.1, which prevents the formation of C5a and C5b, synergized with subtherapeutic doses of CTLA4Ig to significantly prolong the survival of C57BL/6 heart grafts that were transplanted into naive BALB/c recipients. Anti‐C5 mAb treatment limited the induction of donor‐specific IFNγ‐producing T cell alloimmunity without inducing Th2 or Th17 immunity in vivo and inhibited primed T cells from responding to donor antigens in secondary mixed lymphocyte responses. Additional administration of anti‐C5 mAb to the donor prior to graft recovery further prolonged graft survival and concomitantly reduced both the in vivo trafficking of primed T cells into the transplanted allograft and decreased expression of T cell chemoattractant chemokines within the graft. Together these results support the novel concept that C5 blockade can inhibit T cell‐mediated allograft rejection through multiple mechanisms, and suggest that C5 blockade may constitute a viable strategy to prevent and/or treat T cell‐mediated allograft rejection in humans.