吲哚胺2,3双加氧酶在丁酸钠诱导未成熟树突状细胞介导T细胞无能的机制

目的 观察吲哚胺2,3双加氧酶(IDO)在丁酸钠诱导不成熟树突状细胞(DC)抑制T细胞免疫反应中的作用.方法 通过粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素(IL)-4体外诱导入外周血来源的DC,第6天加入丁酸钠和不同促成熟因子继续诱导DC分化,逆转录-聚合酶链反应(RT-PCR)、Real-time PCR检测各组IDO mRNA的表达.混合淋巴细胞反应检测各组刺激T细胞增殖能力,进一步在丁酸钠组和对照组分别加入IDO的阻断剂1-甲基色氨酸(1-MT)后了解对T细胞增殖能力的改变.结果 RT-PCR结果显示丁酸钠组DC IDO mRNA表达量(0.84±0.01)高于对照组(0.55±0.01)及脂多糖(LPS,0.53±0.01)等诱导成熟组.Real-timePCR进一步显示与对照组比较,丁酸钠组IDO mRNA的表达升高了约(32.03±4.01)倍.MLR显示丁酸钠组DC刺激淋巴细胞增殖指数(1.53±1.0)低于LPS(9.87±3.8)等成熟组DC,并且采用IDO的抑制剂1-MT可以降低抑制T细胞增殖的能力.结论 丁酸钠诱导下DC可能通过IDO途径降解细胞微环境中的色氨酸来发挥对T细胞增殖的抑制作用.     

细胞因子信号传导抑制因子-1在树突状细胞介导T细胞分化中的作用

目的 观察细胞因子信号传导抑制因子-1(SOCS1)在不同诱导方法下树突状细胞(DC)的表达及其对T细胞刺激活性的影响.方法 1000 U/ml重组人粒细胞-巨噬细胞集落刺激因子(GM-CSF)和500 U/ml白细胞介素(IL)-4体外诱导的人外周血单个核细胞来源的DC,经1mmol/L丁酸钠、1 mg/L脂多糖(LPS)、细胞因子鸡尾酒法诱导成熟,分别以流式细胞仪、酶联免疫吸附试验(ELISA)、混合淋巴细胞反应(MLR)检测DC表面标志、IL-12、IL-10分泌和刺激淋巴细胞增殖能力.Western blot和实时荧光定量聚合酶链反应(Real-time PCR)分别检测各组SOCS1蛋白水平及mRNA的表达.结果 丁酸钠诱导的不成熟DC的SOCS1蛋白表达最高(1.61±0.22,P＜0.05);其SOCS1 mRNA表达差异倍数(1.58±0.11)显著高于成熟组DC(0.99±0.04、1.27±0.05,P＜0.05).高表达SOCS1的DC刺激淋巴细胞增殖的能力(1.53±1.04),IL-12分泌量(142.79 ±15.61)较成熟DC显著降低(P＜0.05);而IL-10分泌(3.29±0.21)较成熟DC比较显著升高(P＜0.05).结论 SOCS1是调节DC介导的T细胞分化的关键因子.     

不同细胞因子诱导对树突状细胞体外分化的影响

目的 观察不同细胞因子诱导对于体外培养的树突状细胞(DC)免疫刺激活性的影响.方法 通过1000 U/ml人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和500 U/ml白细胞介素(IL)-4体外诱导的人单个核细胞来源的DC,经1000 U/ml肿瘤坏死因子(TNF)-α、1 mg/L脂多糖(LPS)、1 mmol/L丁酸钠、细胞因子鸡尾酒法诱导成熟,分别以流式细胞仪、FITC-Dxtran内吞检测、混合淋巴细胞反应(MLR)、酶联免疫吸附试验(ELISA)检测DC的表面标志、内吞能力、刺激淋巴细胞增殖能力和IL-12分泌的变化.结果 鸡尾酒法诱导下的DC成熟标志显著上调,内吞能力减弱186.74±38.66,刺激淋巴细胞增殖能力增强18.23 ±2.22,并且IL-12的分泌能力增加(656.18±38.52)ng/L,说明其显著促进DC成熟.而丁酸钠诱导下的各项成熟指标均下调,导致DC免疫刺激源性减弱.结论 细胞因子鸡尾酒法是诱导DC成熟的最佳方法;而丁酸钠可以抑制DC成熟,改变DC的免疫状态.

Abstract：

Objective To investigate the immune effect of different cytokines on immature dendritic cells (DC) in vitro. Methods The human monocyte-derived DCs were induced in the presence of recombinant human GM-CSF (rhGM-CSF, 1000 U/ml) and interleukin (IL)-4 (500 U/ml). The effects of tumor necrosis factor (TNF) -α (1000 U/ml) , lipopolysaccharide (LPS) (1 mg/L), the cocktail of cytokines (TNF-α, IL-6, IL-1β, PGE2) , and sodium butyrate (1 mmol/L) on the DCs were detected by flow cytometry (FCM), endocytic activity, T cells stimulatory proliferation capacity, and IL-12 production. Results The cocktail of cytokines could up-regulate the major histocompatibility complex (MHC) class Ⅱ and costimulatory molecules of DCs, decrease the endocytic activity 186. 74 ±38. 66, induce a stage of Tcell stimulation 18. 23 ± 2. 22 and promote the T helper cell type 1-skewing factor IL-12 production (656. 18 ±38. 52) ng/L. But the sodium butyrate induced reverse result on DCs compared to cocktail of cytokines. Conclusion The most effective method for inducing dendritic cells maturation was cocktail of cytokines. The sodium butyrate could inhibit the development of mature DCs, resulting in impaired DC function, and modify the outcome of the subsequent immune response.     

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。     

树突状细胞诱导大鼠肝移植免疫耐受的研究

目的 观察大鼠骨髓来源的树突状细胞(DC)对同种异体肝移植大鼠免疫耐受的影响.方法 用不同细胞因子诱导培养大鼠骨髓来源的成熟DC(mDC)和不成熟DC(imDC),采用形态学观察、表型分析和功能学实验对培养细胞进行鉴定,构建Wistar大鼠和SD大鼠肝移植模型,并于移植前5 d注射到受体大鼠内,研究它们对移植后大鼠肝脏病理及存活时间的影响情况.结果 DC细胞形态不规则,大多数细胞表面有细长树枝状突起,mDC和imDC表面均表达表面分子OX62,而表面分子CD86在imDC表面低表达,在mDC表面高表达.imDC和mDC与淋巴细胞相互作用,发现imDC对淋巴细胞的增殖无明显作用,相反mDC对淋巴细胞的增殖有明显的促进作用.注射imDC的受体大鼠的存活时间显著长于注射mDC的受体大鼠,注射mDC的肝移植大鼠在移植后较早且出现较强的急性排斥反应,而注射mDC的大鼠在移植后16 d才开始出现轻度急性排斥反应.结论 建立了在体外大量扩增大鼠骨髓来源DC的方法,imDC能显著延长受体大鼠肝移植的存活时间.     

不同诱导方法对人外周血树突状细胞体外成熟的影响

目的探讨不同诱导方式对于体外培养的树突状细胞（dendritic cells,DC）免疫刺激活性的影响。方法通过rhGM-CSF和IL-4体外诱导的人外周血单核细胞来源的DC,分别用肿瘤坏死因子α（TNF-α）、脂多糖（LPS）、细胞因子鸡尾酒法诱导成熟,再以流式细胞仪、FITC－Dxtran内吞检测、MLR、ELISA法检测DC的表面标志、内吞能力、刺激淋巴细胞增殖能力和IL－12分泌的变化。结果在不同方法促进DC成熟中,细胞因子鸡尾酒法诱导下的DC成熟状态最佳,CD83指数表达高达84．87％,IL-12的分泌量[（656．18±38．52ng／L）]高于其他各组,且刺激淋巴细胞增殖能力最强（P〈0．05）。结论细胞因子鸡尾酒法是体外诱导DC成熟的最佳方法。     

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。     

丁酸钠增强未成熟树突状细胞表达吲哚胺2,3双加氧酶抑制T细胞增殖

目的 观察丁酸钠诱导的不成熟树突状细胞(DCs)吲哚胺2,3双加氧酶(IDO)的表达及其在抑制T细胞免疫反应中的作用.方法 用重组人粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素(IL)-4诱导人单个核细胞来源的未成熟DCs,6 d后分别加入丁酸钠、脂多糖(LPS)和多细胞因子鸡尾酒组合[肿瘤坏死因子(TNF)-α、IL-6、IL-1β、前列腺腺素E2(PGE2)],24h后收集DCs;流式细胞仪检测Des表型,逆转录-聚合酶链反应(RT-PCR)和实时荧光定量PCR检测IDO mRNA的表达,酶联免疫吸附试验(ELISA)检测IL-12分泌;混合淋巴细胞培养(MLR)检测各组DCs对同种异体T淋巴细胞增殖的影响.结果 丁酸钠诱导的DCs呈现典型未成熟DCs的特征,低表达CD83、CD80和HLA-DR,分泌IL-12水平低.与对照组比较,丁酸钠组和LPS组DCs的IDO mRNA的表达分别升高了(32.03±4.02)倍和(1.01±0.43)倍,而鸡尾酒组则降低(3.31±1.07)倍,差异有统计学意义(P<0.01);丁酸钠诱导的未成熟DCs采用IDO抑制剂1-甲基色氨酸(1-MT)处理后,可以有效刺激T细胞增殖,但其能力仍低于LPS或鸡尾酒法诱导的成熟DCs.结论 丁酸钠可显著增强未成熟DCs的表达IDO,并且IDO过表达在其抑制T细胞增殖中起重要作用.     

大鼠胰腺癌微环境诱导未成熟树突状细胞功能的变化

目的 观察胰腺癌微环境对树突状细胞(DC)成熟的影响及功能变化并探讨胰腺肿瘤细胞免疫逃逸的机制.方法 培养树突状细胞,加入粒细胞巨噬细胞集落刺激因子(rmGM-CSF)40μg/L、白细胞介素(IL)-4 40μg/L,培养到第6天时得到大量的未成熟树突状细胞(imDC),加入大鼠胰腺癌细胞(AR42J cell)培养上清液诱导,流式细胞术检测DC的表面分子CD86、CD80的表达(n=6),观察能否延缓或阻断imDC的成熟及其在脂多糖(LPS)刺激后这种作用能否被逆转.并观察AR42J细胞培养上清诱导的Dc对同种异体混合淋巴细胞增殖.结果 加入胰腺癌癌细胞上清液培养的DC,与正常成熟的DC比较,CD80+CD86+阳性率由(70.88±3.60)%降至(7.15±0.71)%,LPS刺激后DC细胞的表面分子CD80+CD86+表达仍然较低(7.43±1.05)%,表明胰腺癌细胞培养上清液对DC的成熟有阻断作用.AR42J细胞上清诱导培养的imDC组刺激同种异体混合淋巴细胞增殖的强度显著低于正常培养的imDC组刺激同种异体混合淋巴细胞增殖的强度.结论 体外大鼠胰腺癌细胞培养上清液可以诱导DC处于不成熟状态,且这种不成熟状态不容易被逆转.     

大鼠调节性树突状细胞的培养与生物学特性鉴定

目的 培养大鼠调节性树突状细胞(rDC)并探讨其生物学特性.方法 培养3种不同树突状细胞(DC):(1)通过加入1×10-6 mol/L地塞米松(DXM)获得未成熟DC (imDC)；(2)通过加入1 mg/L脂多糖(LPS)活化获得成熟(matDC)；(3)通过1×10-6 mol/L DXM预处理1 mg/LLPS活化获得rDC.流式细胞仪检测各种DC表型,酶联免疫吸附试验(ELISA)法测量细胞因子水平.结果 rDC表现为部分成熟的细胞表型,细胞表面CD40表达较imDC组明显增加,CD86和人类主要组织相容性复合体(MHC)-Ⅱ表达与imDC类似.rDC分泌产生白细胞介素(IL)-12、IL-10水平都显著高于imDC,但IL-12生成上调在很大程度上被DXM预处理抵消,因此,反映了机体免疫反应状况的IL-10/IL-12比值较后者有显著升高(1.15 ±0.33、0.43 ±0.08;P＜0.05).结论 DXM预处理LPS活化可以诱导大鼠骨髓细胞获得rDC,表现为部分成熟的细胞表型,细胞表面CD40表达及分泌IL-10/IL-12比值较imDC组增高.     

抗原负载的树突状细胞体外诱导抗肿瘤免疫的研究

目的 观察抗原负载的树突状细胞(DCs)体外诱导抗肿瘤免疫效应,探讨树突状细胞肿瘤疫苗的制备方法 .方法 以细胞因子粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素(IL)-4体外诱导人单核细胞来源的树突状细胞,第6天加入肝癌细胞BeL-7402的冻融抗原并以联合细胞因子鸡尾酒法[肿瘤坏死因子(TNF-α)+IL-6+IL-1β+前列腺素E2(PGE2)]诱导成熟,对照组仅以鸡尾酒法诱导成熟.24 h后收获DCs以流式细胞仪检测其成熟表型CD80、CD83、CD86和LHA-DR,酶联免疫吸附试验(ELISA)法检测其IL-12的分泌,噻唑蓝(MTT)比色法检测其刺激淋巴细胞增殖活性,乳酸脱氢酶释放实验检测其诱导的免疫效应细胞对肝癌细胞的特异性细胞毒作用.结果 联合细胞因子可诱导的DCs成熟和IL-12的分泌(P＜0.05),成熟的DCs有较强的刺激淋巴细胞增殖能力;抗原负载组DCs可诱导效应细胞对肝癌细胞BeL-7402的特异性杀伤作用(P＜0.05).结论 抗原负载的DCs可体外诱导特异性抗肿瘤免疫效应,提示以抗原负载的树突状细胞作为肿瘤免疫治疗的方法 是可行的.     

Differential effects of sodium butyrate and hexamethylene bisacetamide on growth and secretion of cultured human endocrine tumor cells.

Advanced gastrointestinal endocrine tumors respond poorly to conventional chemotherapy. In this study we examined the effects of two agents that promote cellular differentiation, sodium butyrate and hexamethylene bisacetamide, on the in vitro growth and secretory responses of a human pancreatic carcinoid (BON) and human gastrinoma (PT-2 and PT-SM) cell lines that have been established in our laboratory. We found that both sodium butyrate and hexamethylene bisacetamide strongly inhibited growth of BON, PT-2, and PT-SM cells. With continuous exposure of BON cells to sodium butyrate (2 mmol/L), the doubling time was prolonged, from 60 hours in controls to 156 hours, and saturation density was reduced to 28% that of controls. Hexamethylene bisacetamide (4 mmol/L) reduced saturation density to 37% that of controls in BON cells and prolonged the doubling time, from 60 hours to 103 hours. Antiproliferative effects of similar magnitudes were observed in the gastrinoma cell lines. In contrast, differential effects were produced on amine biosynthesis in BON cells; sodium butyrate stimulated levels of 5-hydroxytryptamine in the cells, whereas hexamethylene bisacetamide caused a profound dose-dependent inhibition of amine biosynthesis. The significant antiproliferative activity of sodium butyrate and hexamethylene bisacetamide and the inhibitory effects of hexamethylene bisacetamide on amine biosynthesis warrant evaluation of these agents or analogues for treatment of metastatic carcinoid and gastrinoma.     

Therapeutic effects of sodium butyrate on glioma cells in vitro and in the rat C6 glioma model

OBJECTIVE: Preliminary in vitro studies have indicated that sodium butyrate inhibits the proliferation of cultured glioma cells and induces cellular differentiation, making it potentially useful as a therapeutic agent for patients with glioblastoma multiforme. The purpose of this study was to expand on the preliminary research by investigating the effects of sodium butyrate on multiple cell lines, explanted cells from glioblastoma tumor specimens, and in vivo in the rat C6 glioma brain tumor model. METHODS: Four malignant glioma cell lines (A-172, T98G, U118MG, and C6) and two primary cell cultures derived from human glioblastoma tumor specimens were treated with 2 mmol/L sodium butyrate for up to 72 hours. Sodium butyrate-induced effects on cell morphology, proliferation, cell cycle distribution, migration, glial fibrillary acidic protein staining, and S100beta protein content were determined. For in vivo studies, a total of 64 male Wistar-Furth rats underwent operations to implant C6 glioma cells stereotactically or were used as controls. The rats were treated with escalating doses of sodium butyrate by microinfusion with Alzet minipumps (Durect Corp., Cupertino, CA). RESULTS: Sodium butyrate treatment in vitro produced changes in morphology and glial fibrillary acidic protein expression indicative of cellular differentiation. In cell lines and explanted cells, sodium butyrate consistently inhibited glioblastoma cell proliferation (to 51 +/- 6% that of controls) and migration (to 46 +/- 17%). Intratumoral infusion of 40 mmol/L sodium butyrate prolonged the survival of Wistar-Furth rats with intracerebral C6 tumors (P = 0.013) without detectable toxicity. CONCLUSION: These data support further consideration of direct interstitial infusion of sodium butyrate in a Phase I clinical study for patients with recurrent glioblastoma multiforme.     

大鼠肝卵圆细胞的分离培养和体外分化研究

目的 观察大鼠肝卵圆细胞的活化，为进一步研究肝卵圆细胞特征建立简单的分离培养方法。方法 利用2-乙酰氨基笏／部分肝切除建立肝卵圆细胞活化的大鼠模型，采用选择性消化法从该模型中分离纯化肝卵圆细胞，并做免疫细胞荧光鉴定。用丁酸钠诱导其分化。结果 成功地建立了肝卵圆细胞活化模型，并从中分离培养了表达OV6、CK19、AFP、白蛋白、c-kit、Thy1、CD45和CD34的卵圆细胞。在丁酸钠刺激下它可向肝细胞分化。结论 利用选择性消化法可以分离到肝卵圆细胞，并且其具有肝干细胞的特征。     

树突状细胞融合瘤诱导抗结肠转移癌免疫应答

目的 检测结肠转移癌细胞SW620和树突状细胞（DC）融合构建的肿瘤疫苗对结肠转移癌细胞SW620及其同源结肠癌细胞SW480的杀伤作用。方法 应用促融合剂50％聚乙二醇（PEG）对SW620细胞和从外周血单个核细胞（PBMC）诱生的DC进行融合,用流式细胞仪（FCM）分析其表型并检测融合效率,电镜及免疫细胞化学观察DC、SW620和融合细胞（DC／SW620）形态;^51Cr释放法检测DC／SW620致敏CTL对SW620及SW480细胞的杀伤作用。结果 从PBMC成功诱生出高表达HLA-ABC、HLA-DR、CD80、CD86、CD83的成熟DC,DC／SW620的融合效率达到27．12％。融合细胞兼具DC与肿瘤细胞的结构特点及免疫表型。^51Cr释放法检测DC／SW620激活的CTL对SW620及SW480的杀伤作用强于各对照组（P〈0．01）。结论 DC与SW620细胞融合体外致敏自体T淋巴细胞能生成抗原特异CTL,对结肠转移癌细胞SW620及同源结肠癌细胞SW480有一定的杀伤作用。     

Dendritic cells exposed to nacystelyn are refractory to maturation and promote the emergence of alloreactive regulatory t cells

BACKGROUND: Drugs blocking dendritic cell (DC) maturation might be useful in transplantation by inhibiting the induction of primary alloimmune responses and promoting the emergence of regulatory T lymphocytes (Treg). We investigated the effects of Nacystelyn (NAL), an N-acetyl-L-cysteine derivative, on human DCs, paying attention to the T-cell responses elicited by NAL-treated DCs in vitro. METHODS: Lipopolysaccharide (LPS) was used to induce the maturation of DCs naturally present in blood or generated from human monocytes cultured in interleukin-4 and granulocyte-macrophage colony-stimulating activity. We first analyzed the consequences of NAL on cytokine production and expression of major histocompatibility complex class II and costimulatory molecules. Monocyte-derived DCs were then used as stimulators in mixed leukocyte cultures with naive CD4 T cells. Cytokine levels were measured in culture supernatants; the phenotype of T cells and their capacity to inhibit the proliferation of third-party T-cell responders was determined at the end of the culture. RESULTS: NAL proved to be a potent inhibitor of DC maturation in whole blood experiments and on monocyte-derived DCs. Alloreactive T cells stimulated with DCs pretreated with LPS in the presence of NAL produced much less interferon-gamma but similar levels of interleukin-13 compared with DCs treated with LPS alone. Immature DCs induced Treg, which was not observed with mature DCs. DCs cultured with LPS in the presence of NAL were as efficient as immature DCs to generate alloreactive T cells with regulatory activity. CONCLUSIONS: NAL is a potent inhibitor of DC maturation, which might be useful to promote allograft acceptance by inducing the differentiation of allospecific Treg.