PD-L1在人胎盘源间充质干细胞对脐血CD8~+T细胞免疫调节中的作用

目的:研究PD-L1在人胎盘源间充质干细胞(Human placenta mesenchymal stem cell,hPMSCs)介导的对脐血CD8+T细胞活化、周期及对IL-17分泌免疫调节中的作用。方法:RT-PCR及FCM检测hPMSCs对PD-L1的表达;应用化学合成的PD-L1 siRNA阻断PD-L1在hPMSCs上的表达;免疫磁珠分选脐血CD8+T细胞;FCM分析阻断PD-L1后,hPMSCs对PHA刺激下CD8+T细胞活化、周期及PMA活化下CD8+T细胞分泌IL-17的影响。结果:hPMSCs高表达PD-L1分子,PD-L1 siRNA能有效阻断hPMSCs对PD-L1的表达;FCM分析结果显示,hPMSCs能够抑制CD8+T细胞对CD69的表达,但阻断PD-L1后,CD69的表达与未阻断组相比无明显变化;与未阻断组相比,处于G0/G1期的CD8+T细胞数量明显减少,处于S期的细胞数量明显增加;在hPMSCs存在条件下,脐血CD8+T细胞对IL-17的分泌明显增加,阻断PD-L1的表达后,IL-17的分泌被进一步上调。结论:PD-L1在hPMSCs上表达能够协同hPMSCs对脐血CD8+T细胞周期的抑制,并且能够抑制hPMSCs上调CD8+T细胞对IL-17的分泌。     

人胎盘源间充质干细胞对脐血CD8~+T细胞活化及周期和IL-17分泌的影响

目的:探讨人胎盘源间充质干细胞(hPMSCs)对脐血CD8+T细胞活化、周期及IL-17分泌的调节作用,为其在临床细胞治疗中的应用提供理论依据。方法:应用消化法分离、培养hPMSCs,体外扩增培养3代后用于实验;应用免疫磁珠法分选脐血CD8+T细胞;应用流式细胞术(FCM)分析hPMSCs对植物血凝素(PHA)刺激下脐血CD8+T细胞早期表型CD25、CD69表达和细胞周期的影响;佛波酯(PMA)刺激下脐血CD8+T细胞对IL-17分泌的影响。结果:hPMSCs体外可使脐血CD8+T细胞滞留于细胞周期的G0/G1期,下调活化脐血CD8+T细胞早期表型CD25、CD69的表达,上调其IL-17的分泌。结论:hPMSCs体外可通过对脐血CD8+T细胞周期的影响抑制其活化,并且上调脐血CD8+T细胞IL-17的分泌。     

原发性胆汁性肝硬化患者共刺激分子B7-H4的检测及临床意义

目的 探讨原发性胆汁性肝硬化(primary biliary cirrhosis,PBC)患者共刺激因子B7-1t4的表达及其与疾病发病机制的关系.方法 分别采用荧光实时定量PCR法(real-time PCR)、酶联免疫吸附试验(ELISA)及流式细胞术(FCM)检测65名PBC患者外周血单个核细胞(PBMC)B7-H4mRNA表达水平、血清IL-2水平以及CD4+、CD8+ T细胞亚群和T细胞表面B7-H4表达百分率,同时监测抗线粒体抗体(anti-mitochondrial antibody,AMA)及临床各项生化指标的关系.结果 (1)PBC患者PBMC B7-144 mRNA水平及T细胞表面B7-H4表达百分率显著低于非PBC肝硬化组及健康对照组(P<0.01).(2)活化72 h后各实验组及对照组IL-2含量及CD4+、CD8+、CD4+ CD8+T淋巴细胞表达水平均低于活化前,以非PBC肝硬化组与健康对照组降低显著(P<0.05);PBC组IL-2含量及CD4+、CD4+ CD8+ T淋巴细胞表达水平高于非PBC肝硬化组与健康对照组(P<0.01).(3)AMA-M2阳性患者血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)及γ-谷氨酰转肽酶(GGT)水平升高,其中ALP及GGT升高显著(P<0.05);AMA-M2阳性患者与阴性患者T细胞表面B7-H4表达百分率差异无统计学意义(P>0.05).结论 共刺激因子B7-H4对PBC患者体内T细胞活化增殖及细胞因子分泌的抑制作用减弱,为研究PBC的发生发展和阐明PBC的发病机制提供了试验资料.     

原发性肝癌组织中B7-H3表达与浸润的CD8~+ T细胞数负相关

目的探讨原发性肝癌组织中B7-H3的表达与CD8+T细胞浸润之间的关系。方法采用免疫组织化学技术分析原发性肝癌组织和正常肝组织中B7-H3的表达、CD8+T细胞的浸润数量,对B7-H3的表达与CD8+T细胞浸润数量的相关性进行统计学分析。结果 63例肝癌患者中B7-H3高表达26例(41.3%),低表达37例(58.7%),总阳性率为90.5%,其中正常肝组织基本不表达B7-H3。B7-H3高表达组CD8+T细胞浸润数量明显低于B7-H3低表达组,CD8+T细胞浸润数量与肝癌组织中B7-H3的表达呈负相关。肝癌组织中B7-H3的表达和CD8+T细胞数量在肝癌患者的临床病理分期、是否有局部淋巴结转移以及远隔器官转移等方面具有显著性差异。结论原发性肝癌组织中高表达B7-H3,与肿瘤组织中CD8+T细胞浸润呈负相关。     

补体C5b-9复合物促进树突状细胞成熟

探讨补体C5b-9复合物对树突状细胞成熟及免疫学功能的影响。rhGM-CSF+rhIL-4体外诱导单核细胞分化为不成熟树突状细胞,体外于不成熟树突状细胞表面用补体蛋白纯品组装CSb-9,37℃,5%CO_2温育96 h,流式分析细胞表型及抗原捕获能力;与同种异体CD4~+和CD8~+T细胞共培养检测其免疫刺激功能;ELISA检测细胞因子分泌。结果显示,亚溶解型C5b-9处理DC表面标志CD83、HLA、CD80、CD86、B7-H1、B7-H3、B7-H4以及BTLA等表达上调;DC分泌IL-12及TNF-α上调,抗原捕获能力降低;C5b-9处理DC刺激CD4~+T细胞活化及分泌IFN-γ、IL-2能力增强。结论:补体C5b-9复合物可以促进树突状细胞成熟,衔接天然免疫和特异性免疫。     

原发性肝癌组织中B7-H3表达与浸润的CD8^+ T细胞数负相关北大核心CSCD

目的探讨原发性肝癌组织中B7-H3的表达与CD8+T细胞浸润之间的关系。方法采用免疫组织化学技术分析原发性肝癌组织和正常肝组织中B7-H3的表达、CD8+T细胞的浸润数量,对B7-H3的表达与CD8+T细胞浸润数量的相关性进行统计学分析。结果 63例肝癌患者中B7-H3高表达26例(41.3%),低表达37例(58.7%),总阳性率为90.5%,其中正常肝组织基本不表达B7-H3。B7-H3高表达组CD8+T细胞浸润数量明显低于B7-H3低表达组,CD8+T细胞浸润数量与肝癌组织中B7-H3的表达呈负相关。肝癌组织中B7-H3的表达和CD8+T细胞数量在肝癌患者的临床病理分期、是否有局部淋巴结转移以及远隔器官转移等方面具有显著性差异。结论原发性肝癌组织中高表达B7-H3,与肿瘤组织中CD8+T细胞浸润呈负相关。     

B7-H3Ig重组蛋白的真核表达及其对T细胞的作用

目的 真核表达人B7-H3Ig重组蛋白,探讨B7-H3信号在T细胞中的作用.方法 重叠PCR方法构建B7-H3Ig重组基因,进而获得重组逆转录病毒载体pGEZ-Term/B7-H3Ig,转染L929细胞建立L929/B7-H3Ig基因转染细胞株,纯化获得B7-H3Ig重组蛋白.体外激发型CD3单克隆抗体(mAb)刺激活化T细胞过程中加入B7-H3Ig,观察其对T细胞增殖以及IL-10与IFN-γ分泌的作用.结果 B7-H3Ig经Protein G柱纯化后纯度＞90％,浓度达0.559 mg/mL.重组蛋白结合实验表明CD3 mAb刺激48 h后T细胞表面B7-H3受体表达达到高峰.B7-H3Ig以剂量依赖方式促进CD3 mAb诱导的T细胞体外增殖与IL-10和IFN-γ分泌.结论 成功建立真核表达人B7-H3Ig基因转染细胞株,B7-H3Ig协同刺激T细胞增殖与IL-10和IFN-γ分泌.     

CD40配基化调节小鼠树突状细胞上B7-H3分子表达的研究

目的 探讨CD40配基化对小鼠骨髓来源树突状细胞上B7-H3分子表达的调节作用及其生物学意义。方法 采用GM-CSF和IL-4联合方案体外诱导小鼠髓系DC，并利用mCD40-CHO和TNF-α分别刺激凋亡肿瘤细胞负载的Dc制备成熟DC；采用间接免疫荧光标记法检测成熟Dc上B7-H3分子的表达；RT-PCR检测B7-H3 mRNA转录水平；混合淋巴细胞反应（MLR）和B7-H3单抗阻断实验分析CD40配基化的DC表面B7-H3分子在T细胞活化中的作用；^3H-TdR掺入试验检测DC对T淋巴细胞的促增殖效应；ELISA测定各组MLR反应和DC培养上清中IFN-γ分泌水平。结果 B7-H3分子在DC不同分化发育阶段均有表达，CD40配基化能显著上调凋亡肿瘤细胞负载的DC中B7-H3表达，TNF-α激发的DC弱表达（P〈0．05）；阻断CD40配基化的DC上B7-H3分子能抑制T细胞增殖和IFN-γ分泌；CD40配基化促进凋亡肿瘤细胞负载的DC分泌IFN-γ量也明显高于TNF-α组（P〈0．05）。结论 体外CD0配基化DC的B7-H3分子上调性表达有助于其刺激T细胞增殖和IFN-γ的产生。     

B7-H4在人子宫颈癌的表达及其与肿瘤内浸润T细胞亚群的相关性研究

目的:研究共刺激分子B7-H4在人子宫颈癌的表达及其与肿瘤内浸润的FOXP3+、CD4+T、CD8+T细胞数量和分泌细胞因子的关系。方法:采用SP免疫组织化学染色检测B7-H4在30例正常人宫颈组织、30例高级别宫颈上皮内瘤变(Cervical intraepithelial neoplasiaⅡ-Ⅲ,CINⅡ-Ⅲ)和67例子宫颈癌的表达;间接免疫荧光双标(Indirect immunofluorescentdouble-staining)观察肿瘤内浸润的FOXP3+、CD4+T、CD8+T细胞数量及其TGF-β1和IFN-γ分泌情况。结果:B7-H4不表达于正常人宫颈上皮,仅在部分瘤变宫颈上皮微弱表达;子宫颈癌B7-H4的阳性表达率为46%(31/67),显著高于正常人宫颈上皮和瘤变宫颈上皮(P<0.01,P<0.05);子宫颈癌B7-H4阳性组病灶内浸润的CD8+T细胞以及分泌IFN-γ的CD8+T数量显著低于B7-H4阴性组(P<0.001,P<0.035);B7-H4的表达与肿瘤内浸润的FOXP3+细胞、CD4+T细胞以及分泌TGF-β1的CD4+T细胞数量无关(P>0.05,P>0.05)。结论:B7-H4在人子宫颈癌细胞异常高表达,并与肿瘤内浸润的CD8+T细胞数量减少以及分泌IFN-γ减少有关,提示B7-H4在抑制肿瘤微环境的细胞免疫中发挥作用。     

B7-H1阻断对CD3AK细胞体外生物学活性的影响

目的: 探讨B7-H1阻断对CD3AK细胞增殖活化及其抗肿瘤免疫效应的影响.方法: 利用CD3单克隆抗体(mAb)刺激健康人外周血淋巴细胞诱导产生CD3AK细胞,然后利用B7-H1阻断型抗体阻断B7-H1通路,3H-TdR渗入法检测阻断后CD3AK细胞的增殖能力,ELISA法检测阻断后CD3AK细胞分泌IFN-γ、 TNF-α和IL-10的水平,同时将CD3AK细胞作用于膀胱肿瘤BIU-87细胞,MTT法检测阻断后CD3AK细胞的杀伤活性.结果: B7-H1阻断后,CD3AK细胞的增殖能力明显增强,体外存活时间明显延长;其分泌IFN-γ、 TNF-α的水平明显提高,而分泌IL-10的水平明显下降;同时CD3AK细胞对BIU-87细胞的杀伤活性亦明显升高.结论: 阻断B7-H1通路可以促进和维持CD3AK细胞的增殖和活化,并增强其抗肿瘤的免疫效应.阻断B7-H1通路将有望成为肿瘤免疫治疗的新策略.     

TWEAK蛋白对肝癌细胞增殖的影响

目的 观察TWEAK蛋白对肝癌细胞系增殖的影响,探讨TWEAK对肝癌细胞增殖的作用.方法构建PAVU6-TWEAK siRNA干涉质粒,转染HepG2和QGY7703肝癌细胞株,MTT法检测抑制TWEAK蛋白表达对肝癌细胞增殖的影响,并于培养体系中加入TWEAK蛋白,检测肝癌细胞增殖活性.结果成功构建PAVU6-TWEAK siRNA干涉质粒,转化肝癌细胞后肝癌细胞增殖受抑.肝癌培养体系中加入TWEAK蛋白后,肝癌细胞增殖活性有所增强.结论 TWEAK蛋白可促进肝癌细胞的增殖活性,抑制TWEAK表达,一定程度上可抑制肝癌细胞增殖.     

Tumor cell and immune cell profiles in primary human glioblastoma: Impact on patient outcome

The distribution and role of tumor‐infiltrating leucocytes in glioblastoma (GBM) remain largely unknown. Here, we investigated the cellular composition of 55 primary (adult) GBM samples by flow cytometry and correlated the tumor immune profile with patient features at diagnosis and outcome. GBM single‐cell suspensions were stained at diagnosis (n = 44) and recurrence following radiotherapy and chemotherapy (n = 11) with a panel of 8‐color monoclonal antibody combinations for the identification and enumeration of (GFAP⁺CD45⁻) tumor and normal astrocytic cells, infiltrating myeloid cells —i.e. microglial and blood‐derived tumor‐associated macrophages (TAM), M1‐like, and M2‐like TAM, neutrophils. and myeloid‐derived suppressor cells (MDSC)— and tumor‐infiltrating lymphocytes (TIL) —i.e. CD3⁺T‐cells and their TCD4⁺, TCD8⁺, TCD4⁻CD8⁻, and (CD25⁺CD127^lo) regulatory (T‐regs) subsets, (CD19⁺CD20⁺) B‐cells, and (CD16⁺) NK‐cells—. Overall, GBM samples consisted of a major population (mean ± 1SD) of tumor and normal astrocytic cells (73% ± 16%) together with a significant but variable fraction of immune cells (24% ± 18%). Within myeloid cells, TAM predominated (13% ± 12%) including both microglial cells (10% ± 11%) and blood‐derived macrophages (3% ± 5%), in addition to a smaller proportion of neutrophils (5% ± 9%) and MDSC (4% ± 8%). Lymphocytes were less represented and mostly included TCD4⁺ (0.5% ± 0.7%) and TCD8⁺ cells (0.6% ± 0.7%), together with lower numbers of TCD4⁻CD8⁻ T‐cells (0.2% ± 0.4%), T‐regs (0.1% ± 0.2%), B‐lymphocytes (0.1% ± 0.2%) and NK‐cells (0.05% ± 0.05%). Overall, three distinct immune profiles were identified: cases with a minor fraction of leucocytes, tumors with a predominance of TAM and neutrophils, and cases with mixed infiltration by TAM, neutrophils, and T‐lymphocytes. Untreated GBM patients with mixed myeloid and lymphoid immune infiltrates showed a significantly shorter patient overall survival versus the other two groups, in the absence of gains of the EGFR gene (p = 0.02). Here we show that immune cell infiltrates are systematically present in GBM, with highly variable levels and immune profiles. Patients with mixed myeloid and T‐lymphoid infiltrates showed a worse outcome.     

把人体皮肤成纤维细胞重编程为干细胞

近年来,关于人体干细胞的研究,特别是将已分化细胞(如人皮肤的成纤维细胞)转化成人体胚胎干细胞的研究取得了突破性进展。在反转录因子OCT3/4、SOX2、C-MYC和KLF4的作用下,人皮肤的成纤维细胞可以转变为多能干细胞,具备了多种干细胞的能力,有望在多种疾病的机制研究和治疗中发挥重要的作用。本文将对这项最新的研究成果进行介绍。     

Lack of effect of LTB4 on human natural killer cell activity and T lymphocyte transformation

Human natural killer (NK) cell activity was not significantly affected by leukotriene (LT) B4 over a wide concentration range (10(-6)-10(-14) M), whether added directly to the NK cell assay or after preincubation of effector cells for 2 h with the drug before addition of Cr labelled K562 target cells. In addition, LTB4 did not affect the kinetics of NK cell mediated cytotoxicity. Addition of LTB4 (10(-6)-10(-10) M) to concanavalin A (Con A) or phytohaemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC) had no significant effect on proliferation measured by tritiated thymidine incorporation, using both optimal and sub-optimal mitogen concentrations. Whilst it is clear that LTB4 is an important mediator of inflammation involving polymorphonuclear (PMN) leukocytes in vivo, it does not affect NK cell or T cell function in vitro.     

人肺腺癌细胞系与支气管上皮细胞系差异蛋白质组学研究

目的:通过对肺腺癌细胞系A549细胞和正常上皮细胞系HBE蛋白组成差异的分析,寻找与肺癌发生、发展相关的差异蛋白,为进一步阐明肺癌发病机制提供更多有价值的信息;为诊断或者预后提供候选分子标记物。方法:运用固相pH梯度双向电泳和基质辅助激光解吸电离飞行时间串联质谱(MALDI/TOF/TOF/MS)分析技术筛选出A549细胞和支气管上皮细胞系HBE间表达水平显著差异的蛋白,并且利用免疫印迹方法对筛选的标记物蛋白进行验证。结果:筛选出21个差异蛋白,涉及到细胞的代谢、蛋白质修饰、细胞运动,通道蛋白和信号转导调控等方面。鉴定并且验证在A549细胞中显著高表达热休克蛋白27(HSPB1)。结论:肺腺癌细胞系与正常支气管上皮细胞系蛋白质表达存在显著差异,其中肺腺癌中高表达HSPB1可能在肺腺癌癌变过程中发挥重要作用,本结果为进一步寻找肺腺癌癌变蛋白标志物奠定了基础。     

人巨细胞病毒对体外培养的人涎腺导管上皮细胞周期的影响及其相关机制

目的 研究人巨细胞病毒（HCMV）对人涎腺导管上皮细胞（HSG）细胞周期的影响及其相关机制.方法 体外培养HSG;用RT-PCR及nest-RT-PCR法检测感染HCMV的HSG中立即早期基因（ie1/ie2）的转录;用流式细胞仪检测HCMV对HSG细胞周期的影响;用蛋白印迹技术检测HCMV感染细胞中周期素D1的表达.结果 感染HCMV的HSG中可检测到HCMV ie1/ie2的转录;HCMV通过影响细胞周期的G1/S关卡,使HSG阻滞于G1期;感染HCMV的HSG周期素D1表达下调.结论 在体外HCMV通过作用于细胞周期的G1/S关卡及下调周期素D1抑制涎腺导管上皮细胞的增殖.     

Plaque-forming cell response of pokeweed mitogen stimulated frozen human lymphocytes

The plaque-forming cell (PFC) response of pokeweed mitogen (PWM) activated fresh or frozen human peripheral blood lymphocytes (PBL) was monitored by the protein A hemolytic plaque assay. Fresh PBL and PBL stored in nitrogen for 7 days to 8 years were tested. On the basis of our preliminary results we conclude that cryopreserved cells secrete the same amounts of immunoglobulin (Ig) as freshly prepared cells.     

Defective antigen-presenting cell function in human neonates

Immaturity of the immune system has been suggested as an underlying factor for the high rate of morbidity and mortality from infections in newborns. Functional impairment of neonatal T cells is frequently quoted as the main underlying mechanism for such immaturity. However, recent studies suggest that neonatal antigen-presenting cells (APCs) also exhibit functional alterations, which could lead to secondary defects of adaptive T-cell responses. In this review, we summarize what is known on the functionality of APC at birth and during early childhood. Compared to adults, neonatal APCs display markers of immaturity and produce low levels of cytokines. Multiple factors could be involved in neonatal APC alteration, such as intrinsic immaturity, defective interaction between APCs and T cells and regulatory T-cell-mediated inhibition. Characterization of the relative contribution of each mechanism is clearly needed to better understand the functional capability of the neonatal immune system.     

Aberrant expression of alpha-Gal on primary human endothelium does not confer susceptibility to NK cell cytotoxicity or increased NK cell adhesion

The contribution of Gal alpha 1,3Gal (alpha-Gal) to cell-mediated organ xenograft rejection is controversial. We have used recombinant lentiviruses encoding a porcine alpha 1,3 galactosyltransferase (alpha 1,3GalT) to obtain alpha-Gal-expressing primary human aortic endothelial cells (HAEC) at a frequency of 70-90%. These cells were compared to non-transduced and mock-transduced HAEC with regard to their susceptibility to human NK cell-mediated lysis, ability to stimulate IFN-gamma production by NK cells, and support of NK cell adhesion under static and dynamic conditions. Using green fluorescent protein (GFP) as a reporter gene, it was shown that the frequency of green fluorescent HAEC increased until day 5 post-transduction, and at a multiplicity of infection of 2.5, it reached 98%. Lentiviral transduction did not result in activation of HAEC, and transduced HAEC responded as expected to TNF-alpha and IFN-gamma stimulation. No differences were detected between non-alpha-Gal- and alpha-Gal-expressing HAEC in terms of their susceptibility to NK cell-mediated lysis, ability to stimulate IFN-gamma production by NK cells, or ability to support NK cell adhesion under static and dynamic conditions. In conclusion, these data argue against an important role for the alpha-Gal epitope in the direct interaction between endothelium and NK cells and prove that recombinant lentiviruses are efficient gene carriers for primary human endothelial cells.