IDO修饰的成纤维细胞培养上清对小鼠T淋巴细胞增殖的影响

目的：检测吲哚胺2—3双加氧酶（IDO）基因修饰的细胞对T淋巴细胞的免疫抑制作用。方法：将pEGFP-NI-mIDO真核表达载体转染至小鼠NIH 3T3成纤维细胞系。体外检测mIDO修饰的成纤维细胞培养液中色氨酸水平的变化；使用CFSE标记技术，检测mIDO修饰的成纤维细胞培养液对ConA刺激的小鼠T淋巴细胞增殖的影响。结果：将pEGFP-NI-mIDO转入NIH 3T3成纤维细胞，流式细胞术（FCM）检测到EGFP的表达；体外实验表明，成纤维细胞培养上清和转染pEGFP—NI的成纤维细胞培养上清可检测到一定量的色氨酸水平，而转染pEGFP-NI-mIDO的成纤维细胞培养上清未检测到色氨酸，提示pEGFP-NI-mIDO转染的成纤维细胞表达了具有活性的mIDO；使用CFSE标记技术，检测mIDO修饰的成纤维细胞培养上清对小鼠T淋巴细胞增殖的影响，发现ConA刺激的IDO组，72h后亲代细胞占各代细胞的百分率明显高于对照组。结论：IDO基因修饰的成纤维细胞可抑制T淋巴细胞的增殖。     

IL-10基因修饰的大鼠树突状细胞表型分析及生物学特性的研究

目的研究IL-10基因修饰后的大鼠树突状细胞(DC)的表型及其生物学特性。方法以含IL-10基因的重组腺病毒载体体外转染大鼠骨髓来源的DC,Western blot测定转染后各组DC中IL-10蛋白的表达,流式细胞仪检测各组DC表面抗原CD83、CD86分子的表达情况,混合淋巴细胞反应法测定各组DC刺激同种异体T细胞增殖的能力。结果 IL-10基因修饰组DC可检测到IL-10高表达,表面抗原CD83、CD86低表达,其刺激T淋巴细胞增殖水平较其他各组低。结论 IL-10基因修饰的DC可有效的表达有功能的IL-10,为研究IL-10修饰的DC诱导同种异体移植免疫耐受奠定了基础。     

脂质体介导的IDO基因转染未成熟树突状细胞对其成熟性及功能的影响

目的:探讨脂质体介导的IDO基因转染对未成熟树突状细胞(Immature dendritic cells,imDCs)的成熟性及功能的影响。方法:从BALB/c小鼠骨髓培养imDCs,形态学观察后用流式细胞术鉴定imDC后,将其分为IDO+-imDC组:重组pEGFP-N1-IDO质粒在脂质体介导下转染imDCs;IDO--imDC组:pEGFP-N1空质粒转染imDCs;imDCs组:imDCs不做特殊处理;mDC组:将培养的imDC用TNF-α诱导成熟。流式细胞术检测IDO基因转染imDC细胞后细胞表型是否发生变化。Western blot检测各组IDO蛋白表达。混合淋巴细胞反应检测各组在体外刺激T淋巴细胞增殖的能力。结果:BALB/c小鼠骨髓培养的细胞表面标志和细胞形态符合imDCs典型特征;Western blot结果显示IDO+-imDC组可见IDO蛋白表达;IDO+-imDC组细胞表面分子CD80、CD86、MHCⅡ表达率分别为(9.4±2.2)%、(8.7±1.1)%、(11.4±2.6)%,imDC组分别为(8.5±1.8)%、(7.5±1.6)%、(10.2±2.1)%,两组比较无显著差异(P均0.05),IDO+-imDC组在体外刺激T淋巴细胞增殖的能力明显低于imDC组(678±90.3vs1199±275.5,P0.01)。结论:脂质体介导的IDO基因转染imDCs能维持细胞于未成熟状态,在MLR中,对T细胞增殖有明显的抑制作用。     

HBV S基因修饰树突状细胞疫苗诱导特异性CTL反应的探讨

目的分析腺病毒载体介导HBVS基因修饰树突状细胞（DCs）能否诱导抗HBV特异性CTL反应。方法制备携带HBVS基因的重组腺病毒，分别转染外周血诱导培养的DCs，观察腺病毒转染DCs效率和DCs中HBV抗原的表达；混合淋巴细胞反应测定HBV抗原基因修饰DCs刺激同种异体T淋巴细胞增殖能力；乳酸脱氢酶释放法检测特异性CTL细胞对HepG2 22．1．5靶细胞的杀伤能力。结果腺病毒载体能够高效介导HBVS基因在DCs中表达，且DC细胞形态完整；HBVS基因修饰DCs具有刺激同种异体T细胞增殖能力，同时能诱导抗HBV特异性CTL反应。结论HBVS基因修饰DCs疫苗具有增强抗HBV特异性CTL效应的能力，可能发展为一种新型抗病毒疫苗。     

淋巴细胞趋化因子增强肝癌树突状细胞融合瘤苗体外免疫作用

目的：观察淋巴细胞趋化因子(lymphotactin, Lptn)基因修饰的肝癌树突状细胞(dendritic cells, DC)融合瘤苗的体外生物学特征和免疫作用。方法： 以重组Lptn基因修饰小鼠骨髓来源的DC，在聚乙二醇(polyethylene glycol，PEG)作用下与H22小鼠肝癌细胞融合，分别以RT-PCR及ELISA方法检测Lptn mRNA及蛋白水平表达，流式细胞仪分析细胞表面免疫分子表达。MTT法检测Lptn基因修饰的肝癌树突状细胞融合瘤苗(DCLptn/H22)对同种异体T淋巴细胞的体外刺激作用。LDH法检测DCLptn/H22融合瘤苗诱导产生的杀伤性T淋巴细胞活性。结果： Lptn基因修饰的DC能分泌较高浓度的淋巴细胞趋化因子，并且具有明显的趋化淋巴细胞功能。DCLptn/H22不但增强融合瘤苗刺激同种异体T淋巴细胞增殖, 而且能增强杀伤性T淋巴细胞活性。结论： 淋巴细胞趋化因子基因修饰能增强融合瘤苗体外免疫刺激作用。     

脂质体介导pIDO-EGFP转染原代培养软骨细胞的初步研究

目的：检测脂质体介导pIDO-EGFP转染原代培养的C57小鼠关节软骨细胞的瞬时表达及转染效率，建立原代培养的小鼠关节软骨细胞转染方法。方法：大肠杆菌中扩增pIDO-EGFP质粒，在最优化条件下通过lipofectamine2000^TM转染试剂将pIDO-EGFP质粒转入原代培养的小鼠关节软骨细胞。应用荧光显微镜和激光共聚焦显微镜观察其转染过程及瞬时表达情况，流式细胞术检测其转染效率。结果：质粒携带的增强型绿色荧光蛋白在转染后24h得到了明显表达，48h后流式细胞术检测其转染效率为36．43％，未影响软骨细胞贴壁过程。结论：经绿色荧光蛋白检测表明，脂质体成功地将IDO基因转染进入原代培养的软骨细胞。转染后的软骨细胞在体外仍能存活，在最优化的条件下能达到良好的瞬时转染效率，为组织工程化软骨细胞基因导入和基因修饰提供了思路。     

增强型绿色荧光蛋白基因转染对原代培养的人软骨细胞细胞周期的影响

目的:观察转染增强型绿色荧光蛋白 (EGFP) 基因后原代培养的人鼻中隔软骨细胞的细胞周期的变化,建立原代培养的人鼻中隔软骨细胞的示踪方法。方法:在大肠杆菌中扩增pEGFP-N1 质粒,通过Amaxa细胞核转染仪将pEGFP-N1质粒转入原代培养的人鼻中隔软骨细胞,应用激光共聚焦显微镜观察其转染过程及瞬时表达情况,流式细胞仪检测其转染效率和细胞周期的变化。结果:增强型绿色荧光蛋白基因在转染24 h后得到了明显表达,48 h后流式细胞仪检测其表达率为35.37％,细胞周期没有明显的改变,且未影响软骨细胞的贴壁过程。结论:经pEGFP-N1质粒转染的原代培养的人鼻中隔软骨细胞仍能在体外存活,对软骨细胞的生长没有明显的影响,pEGFP-N1是转染原代培养的人鼻中隔软骨细胞较为理想的瞬时表达载体,也是组织工程化软骨形成过程的良好示踪剂。     

骨髓间充质干细胞免疫调节机制的实验研究

目的:研究骨髓间充质干细胞(MSCs)上的吲哚胺2,3-过氧化酶(IDO)活性对异基因T淋巴细胞增殖的影响,进而探讨MSCs的免疫调节机制。方法:从人骨髓中分离培养MSCs,并通过其形态的均一性及流式细胞术检测表面标志以鉴定其纯度。经200U/ml IFN-γ作用后的MSCs以RT-PCR和Western blot分别检测IDO mRNA和IDO蛋白表达。有或无1-甲基色氨酸(1-MT)存在下,分别以1×105、5×104、1×104、5×103个MSCs与5×105个外周血异基因T淋巴细胞(MSCs∶T数量比为1∶5、1∶10、1∶50、1∶100)建立混合淋巴细胞培养体系(MLR),以单独培养T淋巴细胞为对照组,MTT法检测T淋巴细胞增殖率,反相高效液相色谱法检测各MLR体系上清中IDO活性。结果:IFN-γ作用后的MSCs出现IDO mRNA和IDO蛋白的表达。当MSCs为1×105、5×104、1×104(即MSCs∶T为1∶5、1∶10、1∶50)时,与对照组比较,T淋巴细胞的增殖率显著降低,IDO活性显著升高;加入1-MT后,T淋巴细胞的增殖率及IDO活性均恢复。当MSCs为5×103(即MSCs∶T为1∶100)时,与对照组比较,T淋巴细胞的增殖率轻度升高(P>0.05),IDO活性无明显变化(P>0.05)。结论:MSCs通过IDO活性在体外发挥免疫抑制作用。     

Survivin基因修饰DCs疫苗激发抗喉癌作用的实验研究

目的构建survivin基因修饰DCs疫苗,并观察其生物学特性及其对喉癌治疗作用。方法通过同源重组构建人全长survivin-腺病毒载体(pAd-survivin),并转染未成熟DCs,诱导培养获取成熟DCs,激活T淋巴细胞的增殖;观察DCs疫苗在体内外的生物学活性。结果重组pAd-sur修饰DCs疫苗能显著刺激T淋巴细胞增殖;pAd-sur-DCs能促进T淋巴细胞IFN-γ分泌增加,与对照组比较差异具有显著意义;疫苗对体外细胞杀伤率51.46%,与对照组比较,差异具有显著意义;对移植瘤细胞凋亡率为54.9%,坏死率为21.87%,与对照组比较,差异具有显著意义。结论经survivin基因修饰后,DCs表面分子的表达率较未经基因修饰DCs显著增高,具备更强的刺激T淋巴细胞增殖,促进IFN-γ分泌;在体内外具有促进杀伤喉癌细胞的能力。     

骨髓间质干细胞吲哚胺2，3-双加氧酶活性对T淋巴细胞的影响(英文)

目的: 探讨骨髓间质干细胞（MSCs）吲哚胺2，3-双加氧酶（IDO）活性对抑制T淋巴细胞应答反应的影响。方法: 从人骨髓中分离培养间质干细胞,通过其形态特点、表面标志及多向分化能力检测进行鉴定。以浓度为2×105 U/L的 IFN-γ对分离的MSCs诱导18 h，检测MSCs上IDO mRNA和IDO蛋白表达。将经过IFN-γ 2×105 U/L诱导的MSCs预先接种在培养板中，再建立混合淋巴细胞培养(MLR)体系，利用MTT法检测T淋巴细胞增殖率，并用反相高效液相色谱法检测IDO活性。结果: IFN-γ能诱导MSCs上IDO mRNA和IDO蛋白的表达；MSCs的IDO活性抑制MLR体系中T淋巴细胞增殖率。结论： 经IFN-γ刺激后的MSCs在体外可抑制异体T淋巴细胞的免疫应答，IDO活性参与了这种免疫抑制作用的发挥。     

Indoleamine 2,3-dioxygenase regulates T cell activity through Vav1/Rac pathway

The immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) suppresses T-cell responses and promotes immune tolerance in tumor resistance. A previous study determined that IDO inhibits Vav1 mRNA expression and the activation of Vav1 and its downstream targets in T cells in the guanine exchange factor (GEF)-independent pathway. The current study aims to determine whether IDO induces T-cell immunosuppression through Vav1/Rac signaling pathway, which is a GEF-dependent pathway. The correlation between Vav1 mRNA expressions in T cells of tumor infiltrating lymphocytes and the levels of IDO expression in lung cancer tissues from lung cancer patients was detected. HEK293 cells were stably transfected with human IDO (HEK293-IDO). T cells were isolated from human blood. HEK293-IDO cells were co-incubated with T cells in the presence or absence of an anti-CD3 antibody to activate T cell receptor (TCR) and/or 1-methyl-l-tryptophan (1-MT) to inhibit IDO activity. The early signaling proteins in T-cytoskeleton regulation through Vav1/Rac pathway of T cell were determined. A significant and negative correlation was observed between IDO and Vav1 expression in the tumor microenvironment. IDO, which was produced by HEK293-IDO cells, significantly inhibited the expression of Vav1, which resulted in defective F-actin reorganization. Thus, TCR signaling initiation was damaged. The effects on T-cells induced by the co-culture of HEK293-IDO cells with T cells were attenuated by 1-MT. Results indicate that the inhibitory effects of IDO on T cell immune responses may occur through the down-regulation of Vav1 protein expression and the suppression of Vav1/Rac cascade. These studies provide insight into the mechanisms of immune escape induced by IDO.     

Microparticle-free placentally derived soluble factors downmodulate the response of activated T cells

Soluble placental factors may have immunoregulatory properties and have been demonstrated to inhibit T-lymphocyte proliferation in vitro. On the other hand, placentally derived syncytiotrophoblast microparticles and crude placental homogenates have been demonstrated to inhibit proliferation of mixed lymphocytes in vitro. Because previous studies on placentally derived soluble factors may have been contaminated by the presence of trophoblast-derived microparticles, we prepared microparticle-free placental supernatants. Such supernatants reduced the activation response of T cells, as well as their proliferation and the production of cytokines such as interleukin-2 and interferon gamma, in a dose-dependent manner. This reduction in T-cell proliferation does not appear to be caused by indoleamine 2,3-dioxygenase (IDO) because it was not reversed by the addition of L-tryptophan or an inhibitor of IDO (1-methyl-DL-tryptophan). No evidence was found for the presence of IDO in these supernatants when we used a biochemical assay measuring tryptophan catabolism. We conclude that the placenta produces currently unknown soluble factors that reduce T-cell activation, proliferation, and cytokine production.     

Multiparameter Flow Cytometric Approach for Simultaneous Evaluation of Proliferation and Cytokine‐Secreting Activity in T Cells Responding to Allo‐stimulation

We report a method combining mixed lymphocyte reaction (MLR) using a carboxyfluorescein diacetate succinimidyl ester (CFSE)‐labeling technique, intracellular cytokine immunofluorescence staining (ICIS), and multiparameter flow cytometry for simultaneous determination of proliferation and cytokine‐secreting activity in T cells responding to allo‐stimulation. C57BL/6 (B6) mice and Balb/c mice were used in the experiments. CFSE‐labeled responder splenocytes were cultured with irradiated stimulator splenocytes, followed by ICIS. In both the Balb/c stimulator‐versus‐B6 responder (Balb/c‐vs.‐B6) and the B6‐vs.‐Balb/c allogeneic combinations, interleukin (IL)‐2 secreting cells and interferon (IFN)‐γ secreting cells were identified predominantly in proliferating CD4⁺ and CD8⁺ T cell fractions, respectively. The suitability of this method was proven by demonstrating a close relationship between the values of cytokines in culture supernatants (that were determined by Cytometric Bead Array assay) and indexes for cytokine‐production (that were obtained by multiplying the percentage of cytokine‐producing cells in T cells and mean fluorescence intensity of cytokine‐staining determined by the combined MLR and ICIS).     

Immunosuppressive properties of human amniotic membrane for mixed lymphocyte reaction

The combination of allograft limbal transplantation (ALT) and amniotic membrane transplantation (AMT) has been applied in the treatment of severe ocular surface diseases. The beneficial effect of this combination has been thought to result from possible immunosuppressive ability of amniotic membrane (AM). However, the mechanisms of any such ability remain unknown. In this study, we investigated whether human AM has the ability to suppress allo-reactive T cell responses in vitro. For mixed lymphocyte reaction (MLR), lymphocytes isolated from lymph nodes of C57BL/6 mice (Mls1b, Vbeta6+) were cultured with irradiated splenocytes from DBA/2 mice (Mls1a, Vbeta6-) with or without human AM. For carboxyfluorescein diacetate succinimidyl ester (CFSE) experiments, responder lymph node cells were labelled with a stable intracellular fluorescent dye and cultured with irradiated stimulator cells. The ratio of responder Vbeta6+ T cells was then determined by FACS analysis, and the division profiles of responder Vbeta6+ T cells were analysed by CFSE content. Furthermore, Th1 and Th2 cytokine synthesis by allo-reactive T cells in MLR culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA). Addition of AM to the MLR culture resulted in the significant inhibition of thymidine incorporation compared with control culture lacking AM. The population of responder CD4+Vbeta6+ T cells was significantly reduced in the AM-treated culture in comparison to control. CFSE analysis revealed less division and lower proliferation of responder CD4+Vbeta6+ T cells in cultures with AM than without. In addition, allo-rective T cell synthesis of both Th1 (IL-2 and IFNgamma) and Th2 (IL-6 and IL-10) type cytokine was significantly decreased in the presence of AM. These results indicate that human AM has the ability to suppress allo-reactive T cells in vitro. This inhibitory effect likely contributes to the success of the ALT-AMT combination.     

IDO-EGFP表达载体的构建及其在人软骨细胞中的表达

目的 克隆人吲哚胺双加氧酶(IDO)基因并构建IDO与增强型绿色荧光蛋白(EGFP)融合蛋白哺乳动物细胞表达载体。方法 利用RT-PCR方法从人白细胞克隆IDO基因并构建IDO-EGFP融合蛋白表达载体。通过细胞核转染仪将表达载体转人人软骨细胞进行瞬时表达，以共聚焦显微镜对表达的绿色荧光进行分析。结果 从活化的人白细胞中克隆到IDO基因编码区全长，并构建了IDO-EFFP融合蛋白表达载体。以该表达载体转染原代人软骨细胞，表达的融合蛋白较均匀地分布于整个细胞。结论 成功构建了IDO-EGFP哺乳动物细胞表达载体，为进一步研究IDO奠定了基础。     

Interferon-induced indoleamine 2,3-dioxygenase activity in human mononuclear phagocytes

Interferon (IFN)-induced tryptophan degradation, catalyzed by indoleamine 2,3-dioxygenase (IDO), has been shown to mediate antimicrobial activity in epithelial cells. IDO activity has also been augmented in peripheral blood mononuclear cells (PBMC) treated with IFN or interleukin-2 (IL-2). The effector cells in this population have now been further characterized. PBMCs were isolated from normal donors, separated into monocyte and lymphocyte populations by plastic adherence, treated with IFN or IL-2, and cultivated in medium supplemented with [3H]tryptophan. Culture supernatants were collected after a 48-h incubation and fractionated by high-performance liquid chromatography; radioactivity was determined in fractions corresponding to tryptophan and its metabolites. IFN-gamma and IFN-beta induced IDO activity only in monocytes (plastic-adherent, nonspecific esterase-positive PBMCs). The induction of IDO activity by IL-2 required both monocytes and lymphocytes. Interaction was required between these populations for induction of IDO by IL-2, due to production of IFN-gamma by T lymphocytes, with subsequent IFN-gamma-mediated induction of IDO in monocytes. A number of myeloid cell lines as well as monocyte-derived macrophages were also tested for their ability to be induced to degrade tryptophan in response to IFN treatment. Monocyte-derived macrophages were found to retain their capacity to be induced by IFN-gamma and IFN-beta to degrade tryptophan after differentiation, and to possess seven times more IDO activity per cell than IFN-induced monocytes. However, the presence of lipopolysaccharide (LPS) in the culture medium was required for the maximum induction of IDO activity by IFN-beta. Furthermore, higher concentrations of LPS were sufficient to induce IDO activity in macrophages in the absence of exogenous IFN.     

Multiparameter flow cytometric approach for simultaneous evaluation of proliferation and cytokine-secreting activity in T cells responding to allo-stimulation

We report a method combining mixed lymphocyte reaction (MLR) using a carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeling technique, intracellular cytokine immunofluorescence staining (ICIS), and multiparameter flow cytometry for simultaneous determination of proliferation and cytokine-secreting activity in T cells responding to allo-stimulation. C57BL/6 (B6) mice and Balb/c mice were used in the experiments. CFSE-labeled responder splenocytes were cultured with irradiated stimulator splenocytes, followed by ICIS. In both the Balb/c stimulator-versus-B6 responder (Balb/c-vs.-B6) and the B6-vs.-Balb/c allogeneic combinations, interleukin (IL)-2 secreting cells and interferon (IFN)-gamma secreting cells were identified predominantly in proliferating CD4+ and CD8+ T cell fractions, respectively. The suitability of this method was proven by demonstrating a close relationship between the values of cytokines in culture supernatants (that were determined by Cytometric Bead Array assay) and indexes for cytokine-production (that were obtained by multiplying the percentage of cytokine-producing cells in T cells and mean fluorescence intensity of cytokine-staining determined by the combined MLR and ICIS).