间充质干细胞对巨噬细胞活化及其功能影响的实验研究

目的 探讨骨髓间充质干细胞(MSC)对脂多糖(LPS)诱导的巨噬细胞活化的影响.方法 采用贴壁筛选法分离、纯化小鼠骨髓MSC,巯基乙酸钠腹腔注射刺激后收集小鼠腹腔巨噬细胞(MPM).实验分为四组(A组:MPM;B组:MPM+LPS;C组:MPM+LPS+MSC;D组:MPM+LPS+MSC上清),建立共培养体系.在LPS(终浓度1 μg/ml)刺激巨噬细胞18 h后收集细胞培养上清,检测TNF-α、TGF-β和一氧化氮(NO)等细胞因子分泌量的变化,同时在体系中加入大肠杆菌标准菌株ATCC25922,共孵育24 h后瑞特染色检查巨噬细胞吞噬功能的变化.结果 巨噬细胞活化后培养上清中TNF-α和NO的含量明显上升[分别为(147.4±37.1)pg/ml,(59.9±8.7)μmol/L];而MSC存在时,TNF-α显著减少[(97.6±30.3)pg/ml,P=0.032],NO降到(50.9±29.5)μmol/L(P＞0.05);当有MSC上清存在时,TNF-α和NO进一步减少为(58.3±31.5)pg/ml,(-3.4±2.3)μmol/L(P＜0.01).MSC对巨噬细胞活化后的吞噬率及吞噬指数没有影响.结论 MSC可抑制LPS诱导的小鼠腹腔巨噬细胞的活化,而对其吞噬功能没有影响.     

Fn、TPO基因修饰对人骨髓间充质干细胞的影响

目的 观察纤维连接蛋白(Fn)、血小板生成素(TPO)融合基因修饰对人骨髓间充质干细胞(MSC)的影响.方法 构建携带Fn-TPO融合基因的重组逆转录病毒载体,并以其对骨髓MSC进行基因修饰;观察Fn-TPO基因在骨髓MSC中的表达,以及基因修饰后骨髓MSC的体外增殖、黏附造血细胞和分泌TPO的能力;并将脐血CD34+细胞接种到基因修饰后骨髓MSC形成的滋养层,培养7d观察修饰后骨髓MSC对造血细胞体外扩增和集落形成能力的影响.结果 成功构建携带Fn-TPO基因的重组逆转录病毒载体且以该逆转录病毒载体对骨髓MSC进行体外基因修饰;Fn、TPO基因在骨髓MSC内能够正常转录;基因修饰后的骨髓MSC体外增殖能力[(6.92±0.77)×104/ml]与对照组[(7.18±0.89)×104/ml]比较差异无统计学意义(P＞0.05);基因修饰组和对照组黏附造血细胞能力分别为0.188±0.018和0.167±0.017(P＜0.01),分泌TPO能力分别为(7.46±0.59)ng/ml和(5.58±0.37)ng/ml(P＜0.01),细胞分泌TPO的能力不受培养时间的影响,但受细胞生长状态影响;2×104脐血CD34+造血干/祖细胞经基因修饰后骨髓MSC联合必要细胞因子体外扩增7 d,有核细胞数、CD34+细胞比例、BFU-E、CFU-GM及CFU-GEMM分别为(29.9±2.7)×104、(33.3±2.8)%、109.3±4.1/1×104CD34+细胞、163.7±7.1/1×104CD34+细胞、13.3±1.5/1×104CD34+细胞,较对照组明显增加(P＜0.01).结论 Fn-TPO基因修饰能够增强骨髓MSC黏附造血细胞、分泌TPO及支持脐血CD34+细胞扩增的能力.     

杀菌通透性增加蛋白体外抑制革兰阴性细菌脂多糖激活血小板的作用

本研究旨在探索杀菌通透性增加蛋白(bactericidal permeability-increasing protein,BPI)能否抑制G-细菌脂多糖(LPS)激活血小板的作用。取10例健康体检者全血制备富含血小板血浆(PRP,1×108/ml)。实验分为4组:正常血小板组:不作任何处理;LPS组:LPS(10μg/ml)刺激6 h;BPI组:BPI(100μg/ml)处理1 h;BPI+LPS组:BPI(100μg/ml)预孵育1 h后,再接受LPS(10μg/ml)刺激6 h。应用流式细胞术(FCM)检测各组血小板膜Toll样受体-4(TLR-4)的表达,酶联免疫吸附测定法(ELISA)测定PRP上清中细胞因子释放水平,包括肿瘤坏死因子-α(TNF-α)和白介素-6(IL-6)。结果表明,与正常血小板组相比,LPS刺激血小板后血小板膜TLR-4表达及上清中TNF-α和IL-6浓度均明显增高(P<0.001)。在接受BPI预处理后,LPS刺激血小板表达TLR-4及TNF-α和IL-6的作用明显下降,但仍高于正常血小板组。BPI单独刺激血小板不引起血小板TLR-4表达增高及细胞因子水平改变。结论:BPI能够抑制LPS诱导的血小板活化。     

血管紧张素Ⅱ对急性肺损伤大鼠肺水通道蛋白1表达的影响

目的 观察血管紧张素Ⅱ(AngⅡ)对急性肺损伤(ALI)大鼠肺水通道蛋白1(AQP1)表达的影响及在肺水肿形成中的作用.方法 按随机数字表法将40只SD大鼠分为假手术组、模型组、AngⅡ受体阻滞剂预处理组及治疗组,每组10只.采用失血性休克-内毒素二次打击建立大鼠ALI模型.预处理组静脉注射脂多糖(LPS)前30 min注射AngⅡ受体阻滞剂30 μg/kg,注射LPS后30 min注射30 μg/kg生理盐水;治疗组注射LPS前30 min注射30 μg/kg生理盐水,注射LPS后30 min再注射AngⅡ受体阻滞剂30 μg/kg;模型组注射LPS前、后均给予30 μg/kg生理盐水.制模后6 h处死大鼠,取下腔静脉血,用放射免疫法测定血清肿瘤坏死因子-α(TNF-α)水平;取肺组织,计算湿/干重(W/D)比值,用放射免疫法测定肺组织AngⅡ表达,用逆转录-聚合酶链反应测定AQP1 mRNA表达.结果 与假手术组相比,ALI大鼠血清TNF-α水平及肺组织W/D比值、AngⅡ表达明显增加,AQP1 mRNA表达明显减少.预处理组及治疗组血清TNF-α水平(μg/L)较模型组明显减少(4.79±0.24、5.55±0.36比6.34±0.31,均P<0.05),肺组织W/D比值减小(4.34±0.23、4.85±0.20比5.41±0.26,均P<0.05),AQP1 mRNA表达明显增加(0.854±0.067、0.727±0.081比0.358±0.071,均P<0.05);而AngⅡ表达(ng/g)有所降低(172.19±15.82、202.82±20.47比245.88±26.31),但差异无统计学意义(均P>0.05).AQP1 mRNA表达与AngⅡ表达和肺组织W/D比值均呈负相关(r1=-0.782,r2=-0.726,均P<0.05).结论 ALI时AngⅡ可能直接或通过炎症介质下调肺脏AQP1 mRNA表达,为肺水肿形成的机制之一.     

培养时间对小鼠树突状细胞及其外泌体免疫相关膜蛋白的影响

目的 明确培养时间对树突状细胞及其外泌体免疫相关膜蛋白（CD80、MHC-Ⅰ、MHC-Ⅱ的影响,为今后相关研究提供实验依据。方法 小鼠骨髓细胞经重组粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白介素4(IL-4)诱导分化成树突状细胞,再加入肿瘤坏死因子α(TNF-α)诱导为成熟树突状细胞;超速离心法提取外泌体;蛋白印迹法和Amnis量化成像流式细胞仪对外泌体进行鉴定;Amnis量化成像流式细胞仪检测小鼠树突状细胞以及树突状细胞外泌体膜上免疫相关蛋白CD80、CD11c、MHC-Ⅰ、MHC-Ⅱ的表达情况。结果 体外培养第5天后,约50%以上的树突状细胞表达CD80、CD11c、MHC-Ⅰ、MHC-Ⅱ,第13天达最高水平,其中CD80阳性率为（97.29±0.63）%,CD11c阳性率为（92.31±1.18）%,MHC-Ⅰ阳性率为（97.91±0.49）%,MHC-Ⅱ阳性率为（97.91±0.49）%,差异具有统计学意义（P <0.001）。第13天后逐渐减少,至第30天,仍有约80%的树突状细胞表达MHC-Ⅰ和MHC-Ⅱ免疫分子。外泌体膜上CD80、CD11c、和MHC-Ⅱ表达...     

血清TNF-α、TGF-β1及T淋巴细胞亚群水平与放射性肺病的临床相关性研究

目的研究放疗前后血清TNF-α、TGF-β1及T淋巴细胞亚群水平与急性放射性肺病的关系。方法收集食管癌和非小细胞肺癌患者60例,采用ELISA法检测放疗前后血清TNF-α、TGF-β1水平,采用流式细胞术检测放疗前后T细胞亚群水平,并评价它们与急性放射性肺病之间的关系。结果 (1)放疗后患者血清TNF-α水平升高,但高损伤组(放射性肺病≥2级)与低损伤组(放射性肺病<2级)TNF-α水平无统计学差异。(2)放疗结束时和放疗结束后3个月高损伤组TGF-β1水平显著高于低损伤组[(13.06±5.24)ng/ml vs.(9.14±4.93)ng/ml,P=0.009;(10.88±4.95)ng/ml vs.(7.02±3.42)ng/ml,P=0.008]。(3)放疗结束时和放疗结束3个月高损伤组总T细胞水平显著低于低损伤组[(56.12±9.88)%vs.(66.88±14.27)%,P=0.007;(59.88±10.02)%vs.(66.98±10.21)%,P=0.019],但CD4+和CD8+T细胞水平均未显现出统计学差异。(4)放疗结束时和放疗结束后3个月高损伤组CD4+CD25+T细胞水平显著高于低损伤组[(17.81±3.87)%vs.(14.51±5.19)%,P=0.022;(16.39±3.78)%vs.(13.05±3.61)%,P=0.003]。(5)CD4+CD25+T细胞水平与TGF-β1水平之间存在一定的正相关性。结论放疗后TGF-β1升高、总T细胞降低及CD4+CD25+T细胞水平升高者,发生放射性肺损伤较明显,且TGF-β1与CD4+CD25+T细胞水平之间存在一定的正相关性。     

脂多糖诱导大鼠主动脉内皮细胞表达血管性血友病因子裂解酶的研究

目的 从剂量-效应和时间-效应关系探讨脂多糖(LPS)对大鼠主动脉内皮细胞(RAECs)血管性血友病因子裂解酶(ADAMTS-13)mRNA和蛋白表达的影响.方法 采用组织贴块法培养Wistar大鼠的RAECs,1周后按1∶3传代至第4～5代,将细胞分为空白对照组和0.01、0.1、1、5 μg/ml LPS刺激组,分别于12、24、48、72 h用半定量逆转录-聚合酶链反应(RT-PCR)测定内皮细胞ADAMTS-13 mRNA表达;用酶联免疫吸附法(ELISA)检测上清液内ADAMTS-13蛋白水平.结果 空白对照组有一定量的ADAMTS-13mRNA和蛋白表达.随着LPS刺激浓度增加和刺激时间延长,ADAMTS-13 mRNA和蛋白表达逐渐下降.与空白对照组(25.22±1.41)比较,0.01μg/ml LPS刺激48 h时ADAMTS-13 mRNA表达(18.78±0.86)即明显下降(P＜0.01);0.1μg/ml和1μg/ml LPS刺激24 h时ADAMTS-13 mRNA表达(23.43±0.63、22.41±0.76)即明显下降(P＜0.05和P＜0.01);5μg/ml LPS刺激12 h时ADAMTS-13 mRNA表达(20.01±2.47)即明显下降(P＜0.01).与空白对照组[(115.76±2.36)ng/ml]比较,0.01 μg/ml LPS刺激12 h时ADAMTS-13蛋白表达[(113.43±1.07)ng/ml]即明显下降(P＜0.05);至5 μg/ml LPS刺激72 h时ADAMTS-13蛋白表达[(7.63±2.64)ng/ml]降至最低(P＜0.01).结论 RAECs有一定量的ADAMTS-13mRNA和蛋白表达;不同浓度LPS刺激内皮细胞不同时间后ADAMTS-13 mRNA和蛋白表达降低,具有剂量依赖性和时间依赖性.     

甘草酸二铵对百草枯诱导肺泡上皮Ⅱ型细胞高迁移率族蛋白1的影响

目的了解甘草酸二铵(DG)对百草枯(PQ)诱导肺泡上皮Ⅱ型细胞(alveolar epithelial cellⅡ,AECⅡ)中高迁移率族蛋白1(high mobility group box 1,HMGB1)的影响。方法 AECⅡ用含10%胎牛血清的RPMI 1640培养基,并加入青霉素100 U/ml、链霉素100μg/L,置于37℃含5%CO2的培养箱中培养。将培养后的细胞分为3组,空白对照组(NS组)不采用任何药物干预培养24 h;模型组(PQ组):以1000μmol/L PQ培养24 h;DG预治疗组(DG组):先以0.6 mg/ml DG培养2 h,再以0.6 mg/ml DG+1000μmol/L PQ培养24 h。采用MTT法测定DG对PQ诱导下AECⅡ的增殖作用;采用酶联免疫吸附法检测3组细胞中的HMGB1、Toll样受体4(TLR-4)、髓样分化因子88(My D88)、细胞核因子-κB(NF-κB)、肿瘤坏死因子(TNF)-α含量;采用实时荧光定量聚合酶链式反应测定3组细胞中HMGB1、TLR-4、My D88、NF-κB P65 mRNA表达。结果在1000μmol/L PQ诱导下,AECⅡ以0.6 mg/ml DG干预24 h时生存率最高,以0 mg/ml DG干预生存率最低。与NS组比较,PQ、DG组HMGB1、TLR-4、My D88、NF-κB P65、TNF-α水平均明显升高,且DG组升高程度低于PQ组,差异均有统计学意义(P0.01);与NS组比较,PQ组、DG组HMGB1、TLR-4、My D88、NF-κB P65 mRNA表达均升高,且DG组升高程度低于PQ组,差异均有统计学意义(P0.01)。结论 DG可降低HMGB1、TLR-4、My D88、NF-κB、TNF-α水平,减轻PQ诱导的AECⅡ损伤。     

转染CⅡTA基因上调小鼠树突状细胞表面MHC分子和共刺激分子的表达

目的 探讨MHCⅡ类分子反式激活因子(MHC classⅡtransactivator,CⅡTA)基因对树突状细胞(Dendritic cell,DC)表面MHC-Ⅰ/-Ⅱ类分子及共刺激分子CD80、CD86的调节作用,为将树突状细胞应用于生物治疗奠定基础.方法 从小鼠骨髓中大量扩增DC,用脂质体转染法将小鼠CⅡTA(mCⅡTA)基因转入DC中,用流式细胞术观察转染前、后DC表面MHCⅠ-/-Ⅱ类分子及CD80、CD86的表达.结果 转染mCⅡTA基因使DC上MHC-Ⅰ/-Ⅱ分子的表达率由74.2%/66.7%明显上调为93.6%/91.4%;CD80/CD86的表达率由52.3%/60.5%明显上调为89.7%/91.5%(P＜0.05).结论 转入mCⅡTA可使DC表面MHC-Ⅰ/-Ⅱ分子及CD80/CD86的表达率明显上调.本研究为将DC应用于肿瘤的生物治疗奠定了基础.     

高迁移率族蛋白B1对人脐血造血干细胞增殖分化的影响

目的 观察辐射后骨髓基质细胞(MSC)高迁移率族蛋白B1(high mobility group box 1,HMGB1)的释放,研究HMGB1对人脐血造血干细胞(HSC)增殖分化的影响.方法 体外培养人骨髓MSC,利用ELISA方法 检测经12 Gy γ射线照射后培养上清中HMGB1含量的变化.HMGB1与CD34~+的人脐血HSC体外液体共培养6 d,通过流式细胞术检测CD34~+细胞分化指标(CD13、CD14、CD11c、CD41、CD71)的变化.集落形成实验观察HMGB1对HSC增殖分化的影响.结果辐射后,骨髓MSC培养上清中HMGBl含量为(4.3±0.9)ng/ml,较对照组HMGB1含量[(0.4±0.2)ng/ml]明显升高(P＜0.01).脐血CD34~+细胞表面表达HMGB1受体RAGE、TLR2和TLR4.HSC与HMGB1共培养6d后,与对照组比较,红系(CD71)和粒单系(CD13、CD14、CD11c)标记的表达明显增强,分别为CD13(18.4±3.8)%和(32.6±5.9)%、CD14(12.6±2.7)%和(25.4±4.4)%、CD11c(9.8±2.1)%和(20.3±3.9)%、CD71(26.6±4.6)%和(47.1±7.4)%,而巨核系标记CD41的表达[(1.1±0.4)%和(1.3±0.5)%]无明显变化.集落形成实验示共培养14 d后,红系集落、粒-巨噬细胞集落和总集落的生成较对照组明显增多(P＜0.05),联用抗-TLR2和抗-TLR4抗体可部分抑制这一作用.结论 辐射促进骨髓MSC释放HMGB1,胞外HMGB1可以促进HSC的增殖分化.辐射后,骨髓MSC释放的HMGB1与造血恢复或造血重建的关系值得进一步研究.     

Lipopolysaccharide causes atrophy of Peyer's patches and an increased expression of CD28 and B7 costimulatory ligands

Intestinal mucosal dysfunction appears to contribute to infectious complications in critically ill patients. The current study was undertaken to investigate whether endotoxin affects lymphocyte subpopulations and the expression of costimulatory signals in Peyer's patches (PP). Female Balb/c mice were given an intraperitoneal injection of 25 microg LPS and sacrified 24 h or 72 h later to determine total cell yield, lymphocyte subpopulations (B-cells, total T-cells, CD4+- and CD8+-cells), the costimulatory molecules CD28, B7.1 (CD80) and B7.2 (CD86) and the percentage of apoptotic cells in PP and in the spleen as well as small intestinal IgA concentration. Lipopolysaccharide (LPS) challenge caused a significant decrease of total cell yield in PP at both time-points (-50+/-28% and -43+/-25%, respectively; P < 0.001). This decrease was significant for all measured lymphocyte subpopulations. In contrast, total cell yield was increased (P < 0.001) in the spleen 24 h (+52+/-13%) and 72 h (+130+/-22%) after LPS. The decrease of lymphocyte numbers in the PP was accompanied by an increased percentage of lymphocytes expressing costimulatory molecules. In this respect, an increased percentage of CD40+CD80+, CD40+CD86+, and of CD4+CD28+ could be demonstrated after LPS administration. In the spleen, the percentage of CD4+CD28+ was also elevated after LPS bolus, however, the percentage of CD40+CD80+ was reduced, and that of CD40+CD86+ was unaltered. The influence of LPS on apoptosis of lymphocytes was time-dependent. The percentage of apoptotic cells 24 h after LPS was increased in PP (P < 0.01), but was unchanged in the spleen. Seventy-two hours after LPS injection, the percentage of apoptotic cells returned to normal in PP. Luminal IgA levels remained unchanged after LPS challenge. In conclusion, our data show that LPS causes atrophy of PP which seems to be counterregulated by an enhanced expression of costimulatory molecules.     

Quantitative expression of Toll-like receptor-2, -4, and -9 in dendritic cells generated from blasts of patients with acute myeloid leukemia

BACKGROUND: Dendritic cells (DCs) generated from leukemic blasts constitute a promising tool in immunotherapy for acute myeloid leukemia patients (AML-DCs), because AML-DCs express human leukocyte antigens and costimulatory molecules such as CD40, CD80, and CD86 at a higher level than leukemic blasts. Potentiation of AML-DC vaccine might become feasible by the addition of adjuvants such as lipopolysaccharides (LPS) or CPG-rich oligodeoxyribonucleotides binding to Toll-like receptors (TLR) and inducing a stronger Type 1 T-cell response. STUDY DESIGN AND METHODS: mRNA and protein expression of TLR-2, -4, and -9 were analyzed with quantitative real-time polymerase chain reaction, Western blot, and flow cytometry for mature monocyte-derived DCs generated from 14 AML patients versus 14 healthy volunteers (HV-DCs), and the response of the AML- and HV-DCs to different microbial TLR ligands was determined by enzyme-linked immunosorbent assay for the proinflammatory cytokines tumor necrosis factor (TNF)-alpha, inducible protein (Ip)-10, and interleukin (IL)-6. RESULTS: AML-DCs and HV-DCs strongly expressed TLR-2 and TLR-4, while TLR-9 was expressed at a lower level in both groups. There was no significant difference in TLR expression between the two groups of AML-DCs and HV-DCs. In accordance with the TLR expression levels, DCs generated from both AML patients and HVs responded to the known microbial ligands peptidoglycan (PGN) and lipoteichoic acid for TLR-2 and LPS as ligand for TLR-4, by producing TNF-alpha and IL-6. A response to the ODNs 2006 and 2216 binding to TLR-9 was only detected in AML-DCs. CONCLUSION: Microbial ligands like ODNs and LPS constitute promising adjuvants for enhancing (AML-) DC vaccines.     

Modulation of toll-like receptor 4 expression on human monocytes by tumor necrosis factor and interleukin-6: tumor necrosis factor evokes lipopolysaccharide hyporesponsiveness,whereas interleukin-6 enhances lipopolysaccharide activity

Toll-like receptors (TLR) play a pivotal role in the innate immune response, and the expression levels of these receptors may reflect the sensitivity of immune cells to infections. The binding of lipopolysaccharide (LPS) to TLR-4 triggers human monocytes to produce cytokines, which play a dominant role in the inflammatory response, as can be observed during sepsis and after polytrauma. Here, we evaluated TLR-4 expression of isolated monocytes in the presence of tumor necrosis factor (TNF)-alpha, interleukin (IL) 6, IL-8, and IL-10, and we investigated cellular activation of this treatment. TNF-alpha significantly down-regulated TLR-4 mRNA expression after 6 h (100% vs. 38.5% +/- 4%; P < 0.05). This down-regulation was followed by a dose- and time-dependent diminished expression of TLR-4 surface protein (100% vs. 8.0% +/- 5%; P < 0.01). Forty-eight hours after TNF-alpha treatment, a reduced nuclear factor (NF)-kappaB translocation and a diminished IL-6 secretion after LPS stimulation were found (100% vs. 42.0% +/- 23%; P < 0.05). In contrast, IL-6 incubation upregulated TLR-4 cell surface protein (100% vs. 165.8% +/- 24%; P < 0.05) and increased the ability to activate NF-kappaB and AP-1 after LPS stimulation. Stimulation with IL-8 or IL-10 had no significant effects. We conclude that not only LPS but also TNF-alpha and IL-6 have the potency to regulate the immune response via TLR-4. Down-regulation of TLR-4 by TNF-alpha is associated with LPS hyporeactivity for NF-kappaB formation, whereas upregulation of TLR-4 via IL-6 can increase the responsiveness of mononuclear phagocytes.     

Increased expression of toll-like receptor-2 and -4 on leukocytes from patients with sepsis

The reduced responsiveness of monocytes or granulocytes toward endotoxin (endotoxin tolerance) during sepsis may depend on Toll-like receptors (TLR). The expression of TLR-2 and TLR-4 was measured on neutrophils (PMN) and monocytes from patients with sepsis (n = 21) or healthy controls (n = 12). Leukocytes (1 x 10/mL) were incubated at 37 degrees C with or without a TLR-4 (LPS 1 microg/mL) or a TLR-2 ligand (MALP-2 2 nM). Surface expression of TLR-2 and TLR-4 at 0, 4, and 16 h was determined in FACS after staining with specific antibodies. The release of IL-8 and TNF-alpha was measured by ELISA. Freshly isolated PMN from patients with sepsis exhibited significantly (P < 0.05) higher mean fluorescence for TLR-2 (78.0 +/- 18.6) and TLR-4 (11.4 +/- 2.3) than controls (12.8 +/- 2.2 and 2.3 +/- 0.4). Similarly, monocytes from patients exhibited higher TLR-2 and TLR-4 expression (300.8 +/- 40.6 and 92.7 +/- 12.1) than cells from controls (149.5 +/- 27.1 and 52.2 +/- 7.6). In patients with sepsis, expression of TLR-2 and TLR-4 on PMN increased during 16 h of incubation (106.2 +/- 22.1 and 34.5 +/- 5.3), whereas it remained unchanged in controls (19.3 +/- 6.1 and 5.4 +/- 1.9). Incubation with LPS or MALP-2 had no effect on TLR-4 or TLR-2 expression in cells from either controls or patients. Despite increased TLR expression in cells from patients with sepsis, the endotoxin-induced release of TNF-alpha and IL-8 was indistinguishable from that in controls. Therefore, the endotoxin tolerance seen in patients with sepsis does not depend solely on TLR-2 or TLR-4 expression, and other mechanisms must be involved.     

特发性血小板减少性紫癜患者外周血淋巴细胞共刺激分子的表达及白介素18的作用

本研究探讨特发性血小板减少性紫癜（ITP）患者外周血淋巴细胞共刺激分子CD80、CD86及其配体CD28的表达水平及白介素18的作用。应用免疫荧光标记和流式细胞技术检测34例ITP患者和34名正常人外周血淋巴细胞共刺激分子CD80、CD86及其配体CD28的表达,酶联免疫（ELISA）法测定血浆IL-18。结果表明：ITP患者外周血淋巴细胞CD80、CD86表达率（4.21±2.27%,7.19±5.16%）均明显高于正常对照组（2.34±0.87%,4.08±1.96%,P〈0.01）。ITP组血浆IL-18含量为（538.31±111.33）pg/ml,正常对照组血浆IL-18含量为（489.44±49.07）pg/ml。IL-18含量与血小板数量呈显著性负相关（r=-0.395,P〈0.05）。结论：ITP患者CD80和CD86共刺激分子过度表达,患者血浆中IL-18水平比正常对照组明显升高（P〈0.05）,共刺激分子CD80、CD86与ITP发病密切相关,IL-18在ITP发病机理中起重要作用。     

Decreased response to recall antigens is associated with depressed costimulatory receptor expression in septic critically ill patients

Anti-inflammatory substances are released during septic shock that modulate monocyte function. Decreased monocyte responsiveness to bacterial toxins and decreased expression of human-leukocyte-associated antigen-DR (HLA-DR) have been reported during septic shock and critical illness. Impaired antigen presentation has been inferred from these observations but has not been demonstrated. We assessed antigen presentation and costimulatory molecule expression in 12 age-matched control subjects, 10 noninfected critically ill patients (CINS), and 17 critically ill patients with sepsis (CIS). Antigen presentation was assessed by using in vitro lymphocyte 5-bromo-2-deoxyuridine (BrdU) incorporation in response to tetanus toxoid. The expression of HLA-DR and the costimulatory molecules CD28, CD86, and CTLA-4 was assessed by flow cytometry. Serum interleukin-10 (IL-10) was also measured by enzyme-linked immunosorbent assay. Serum IL-10 levels were significantly elevated in CIS patients (91 +/- 38 pg/mL) as compared with levels in control subjects (5 +/- 4 pg/mL)(P < .05). Lymphocyte BrdU incorporation increased by 710% +/- 243% in control subjects but by only 144% +/- 62% in CIS patients and 76% +/- 31% in CINS patients (P < .01 vs control). Monocyte HLA-DR expression, monocyte CD86 expression, and lymphocyte CD28 expression were significantly decreased in CIS patients (P < .01) as compared with control subjects. Conversely, lymphocyte CTLA-4 expression was significantly increased in CIS patients (P < .05 vs control). Monocyte CD86 expression was also significantly decreased in CINS patients as compared with control subjects. These data indicate that antigen presentation is decreased in critically ill patients with sepsis. This appears in part related to decreased expression of HLA-DR and the costimulatory molecules CD86 and CD28. Increased expression of the negative signal receptor CTLA-4 may also impair antigen presentation in patients with sepsis.     

重型再生障碍性贫血患者骨髓中辅助性T细胞亚群数量及功能的变化

目的　研究重型再生障碍性贫血 (SAA)患者在免疫抑制治疗 (IST)恢复前后骨髓中辅助性T细胞 (Th细胞 )亚群改变情况及与造血功能的关系。方法　以流式细胞仪测定 2 4例发病期、15例恢复期SAA患者及 16名正常对照者骨髓中Th细胞亚群及CD3 CD8 细胞改变情况 ;以放射免疫法测定 2 0例发病期SAA患者、12例IST后恢复期患者及 16名正常对照者血清中TNF α、IL 4水平 ;评价Th1与CD3 CD8 细胞、TNF α的相关关系 ;评价Th1、CD3 CD8 细胞、TNF α、IL 4及Th1/Th2平衡与网织红细胞、中性粒细胞绝对值的相关关系。结果　正常对照组骨髓中Th1细胞、Th2细胞百分率及Th1/Th2比值分别为 (0 .4 2± 0 .30 ) %、(0 .2 4± 0 .17) %、1.5 7± 0 .93;发病期SAA分别为 (4 .87± 2 .6 4 ) %、(0 .4 1±0 .2 6 ) %、2 1.2 2± 5 .0 7,均显著多于正常对照 (P <0 .0 1,P <0 .0 5 ,P <0 .0 1) ,恢复期分别为 (0 .5 3± 0 .2 2 ) %、(0 .4 4± 0 .15 ) %、1.38± 0 .4 5 ,与正常对照组相当 (P均 >0 .0 5 ) ;CD3 CD8 细胞亦由发病期的(32 .32± 18.6 9) %显著下降为 (13.76± 2 .96 ) % (P <0 .0 1) ;SAA发病期血清中TNF α、IL 4为 (4 .2 9± 3.15 ) μg/L、(1.2 4± 0 .73) μg/L ,高于正常对照组的 (1.2 1± 1.16 ) μg/L     

The linkage of innate to adaptive immunity via maturing dendritic cells in vivo requires CD40 ligation in addition to antigen presentation and CD80/86 costimulation

Dendritic cell (DC) maturation is an innate response that leads to adaptive immunity to coadministered proteins. To begin to identify underlying mechanisms in intact lymphoid tissues, we studied alpha-galactosylceramide. This glycolipid activates innate Valpha14(+) natural killer T cell (NKT) lymphocytes, which drive DC maturation and T cell responses to ovalbumin antigen. Hours after giving glycolipid i.v., tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were released primarily by DCs. These cytokines induced rapid surface remodeling of DCs, including increased CD80/86 costimulatory molecules. Surprisingly, DCs from CD40(-/-) and CD40L(-/-) mice did not elicit CD4(+) and CD8(+) T cell immunity, even though the DCs exhibited presented ovalbumin on major histocompatibility complex class I and II products and expressed high levels of CD80/86. Likewise, an injection of TNF-alpha up-regulated CD80/86 on DCs, but CD40 was required for immunity. CD40 was needed for DC interleukin (IL)-12 production, but IL-12p40(-/-) mice generated normal ovalbumin-specific responses. Therefore, the link between innate and adaptive immunity via splenic DCs and innate NKT cells has several components under distinct controls: antigen presentation in the steady state, increases in costimulatory molecules dependent on inflammatory cytokines, and a distinct CD40/CD40L signal that functions together with antigen presentation ("signal one") and costimulation ("signal two") to generate functioning CD4(+) T helper cell 1 and CD8(+) cytolytic T lymphocytes.     

骨髓间充质干细胞对体外分化的原态(naive)T细胞分泌细胞因子的影响

目的观察骨髓间充质干细胞(MSC)对原态(naive)T细胞的反应,探讨其免疫调节机制。方法传代培养3代的MSC经60Co照射后与脐血CD34+细胞分化产生的原态T细胞共孵育1周,用ELISA方法检测培养上清细胞因子的变化。结果孵育7d后,与MSC共孵育后T细胞数量明显增加,为(9.15±0.68)×105/孔,而原态T细胞单独培养组为(4.87±1.33)×105/孔(P<0.05);ELISA检测共孵育组IFNγ分泌减少为(1.147±0.181)pg/ml,而原态T细胞单独培养组为(4.897±0.189)pg/ml、共孵育组IL2分泌增加为(16.141±2.729)pg/ml,而原态T细胞单独培养组为(2.551±0.460)pg/ml,未检测到IL4和IL10。结论MSC有一定的抗原性,与MSC共孵育导致原态T淋巴细胞增殖,MSC可能通过直接或间接抑制T细胞产生IFN-γ发挥免疫抑制作用。为进一步阐明MSC免疫调节机制提出新的线索。     

脐血间充质干细胞的生物学特征及其对造血干/祖细胞体外扩增的支持作用

目的探讨脐血间充质干细胞(MSC)的生物学特征及其对造血干/祖细胞体外扩增的支持作用。方法用液体培养法分离脐血贴壁细胞,采用ELISA方法检测贴壁细胞条件培养液中细胞因子的表达;用流式细胞术分析其免疫表型特征;在成软骨细胞诱导培养条件下诱导细胞分化,并用RTPCR方法检测分化后细胞原胶原Ⅱ型基因的表达。采用分阶段共培养方法观察脐血贴壁细胞对CD34+细胞体外扩增的支持作用。结果脐血单个核细胞纤维样细胞集落形成率为(3.5±0.7)/106。脐血MSC体外至少可以扩增15代。没有分化的脐血MSC表型为CD13、CD29、CD90、CD105、CD166、SH2、SH3和SH4阳性,CD45、CD34和CD14阴性;脐血MSC培养上清中干细胞因子、IL6和肿瘤坏死因子α检测阳性。在成软骨细胞诱导培养基培养条件下,脐血MSC原胶原Ⅱ型基因mRNA表达阳性。脐血MSC与CD34+细胞共掊养14d,CD34+细胞扩增率高于未共培养组4倍。结论脐血MSC具有类似于成体骨髓MSC的特征,对造血干细胞增殖有明显的支持作用。