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。     

表达PD-L1/PD-L2的人胎盘间充质干细胞对外周血T细胞分泌IL-17的拮抗作用

目的 探讨程序性死亡因子配体1(PD-L1)和PD-L2在人胎盘源性间充质干细胞(hPMSCs)上表达对外周血T细胞分泌IL-17的影响.方法 应用酶消化法分离人胎盘间充质干细胞(hPMSCs),并进行表型及分化鉴定;RT-PCR、激光扫描共聚焦显微镜技术(LSCM)及流式细胞术(FCM)检测hPMSCs上PD-L1及PD-L2的表达;应用化学合成的PD-L1 siRNA、PD-L2 siRNA沉默hPMSCs上PD-L1及PD-L2表达;密度梯度离心法分离纯化人外周血T细胞;细胞胞内因子染色法分析沉默PD-L1或PD-L2后hPMSCs对PMA活化的T细胞分泌IL-17的影响.结果 除PD-L1外,hPMSCs高表达PD-L2;siRNA能够有效阻断PD-L1和PD-L2在hPMSCs上的表达;胞内染色结果显示,hPMSCs能够上调T细胞IL-17的分泌,阻断PD-L1或PD-L2后,T细胞IL-17的分泌被进一步上调,且PD-L1和PD-L2具有叠加作用.结论 PD-L1和PD-L2在hPMSCs上表达,可拮抗hPMSCs刺激外周血T细胞分泌IL-17的作用.     

B7-H3在人肝癌细胞株HepG2对外周血CD8+T细胞活化、周期及分泌IL-17调节中的作用

目的 研究B7-H3在人肝癌细胞株HepG2对人外周血CD8+T细胞活化、周期及IL-17分泌等调节中的作用.方法 RT-PCR及FCM检测B7-H3在HepG2细胞上的表达;应用脂质体法将PGPU6/GFP/neo-B7-H3shRNA质粒转入肝癌细胞株HepG2,阻断B7-H3的表达;免疫磁珠分选健康人外周血CD8+T细胞;FCM分析B7-H3分子在HepG2细胞对PHA刺激下CD8+T细胞活化、周期及PMA刺激下CD8+T细胞分泌IL-17调节中的作用.结果 肝癌细胞株HepG2高表达B7-H3分子,PGPU6/GFP/neo-B7-H3 shRNA质粒能有效阻断B7-H3在HepG2细胞上的表达;FCM分析结果显示,肝癌细胞株HepG2对CD8+T细胞活化及周期均有抑制作用;阻断B7-H3的表达后,明显减弱HepG2细胞对CD8+T细胞早期活化表型CD69表达的抑制作用,且能够通过下调CD8+T细胞Go/G1期细胞数量,上调S期细胞数量逆转HepG2细胞对CD8+T细胞周期的阻滞作用;在HepG2存在条件下,CD8+T细胞对IL-17的分泌明显增加,阻断B7-H3的表达后,IL-17的分泌被进一步上调.结论 HepG2细胞高表达B7-H3分子;B7-H3能够协同HepG2细胞对CD8+T细胞活化表型CD69的表达及细胞周期的抑制作用;HepG2细胞上调CD8+T细胞对IL-17的分泌作用,但B7-H3可抑制该上调作用.     

双氢青蒿素对小鼠T细胞的免疫抑制作用

目的:研究双氢青蒿素(DHA)在体外对ConA诱导的小鼠T细胞增殖的影响,探讨其可能的免疫作用机制。方法:加入不同浓度DHA,以多克隆刺激剂刀豆蛋白A(ConA)诱导T细胞活化增殖,用羧基荧光素双醋酸盐琥珀酰亚胺酯(CFDA-SE)染色法分析T细胞增殖情况;利用流式细胞术(FCM)结合双色免疫荧光染色技术检测CD3+T细胞早、中、晚期活化标志CD69、CD25、CD71表达情况;用Fluo-4/AM荧光钙离子探针技术检测细胞内钙离子([Ca2+]i)浓度的变化;以碘化丙锭(PI)染色分析细胞周期分布;运用流式细胞术(FCM)结合三色荧光染色技术检测CD4+CD25highTreg早期活化抗原CD69表达情况。结果:CFDA-SE染色结果显示,DHA能有效抑制ConA诱导的T细胞增殖,并呈时间-剂量依赖性;DHA对ConA刺激的CD3+T细胞CD69、CD25的表达有促进作用,而对CD71的表达有抑制作用,均呈剂量依赖性;DHA单独作用不能引起T细胞[Ca2+]i的升高,在ConA的刺激下能引起[Ca2+]i浓度升高;PI染色流式分析结果显示,DHA阻滞细胞于G0/G1期,阻止细胞进入S期和G2/M期;DHA能够下调CD4+CD25highTreg细胞CD69的表达。结论:DHA对小鼠淋巴细胞的增殖有明显的抑制作用,是一种潜在的免疫抑制剂。     

人骨髓源和胎盘源间充质干细胞对T细胞增殖抑制作用的比较

目的:比较研究人骨髓源和胎盘源间充质干细胞介导的T细胞增殖抑制作用机制。方法:应用流式细胞术(FCM)分别检测B7H4和PDL1在人骨髓源间充质干细胞(HBMSCs)和胎盘源间充质干细胞(HPMSCs)上的表达;应用抗体阻断试验分析B7H4、PDL1分别在HBMSCs和HPMSCs对T细胞增殖及周期影响中的作用。结果:HBMSCs上高表达免疫负性调控分子B7H4,而HPMSCs上高表达免疫负性调控分子PDL1。分别应用B7H4mAb和PDL1mAb阻断,可使HBMSCs和HPMSCs对PHA激发的T细胞增殖抑制作用明显减弱;下调T细胞周期中G0/G1期细胞数量,上调S期细胞数量,明显减弱HBMSCs和HPMSCs对T细胞周期的影响。结论:HBMSCs和HPMSCs可通过表达不同的免疫负性调控分子介导T细胞的增殖抑制作用。     

下调Notch3对小鼠T淋巴细胞体外活化增殖及NF-κB的影响

研究siRNA下调Notch3对小鼠T淋巴细胞体外增殖的影响,并初步探讨其免疫调节机制。从小鼠脾脏分离制备T淋巴细胞悬液;将化学合成的靶向Notch3基因的siRNA在Lipofectamine 2000介导下转染小鼠淋巴细胞;通过Westernblot检测各组细胞中Notch3蛋白水平的变化;以不同浓度的Notch3 siRNA作用于该小鼠T淋巴细胞模型,流式细胞术检测CD3~+T细胞早期活化标志CD69分子的表达;MTT检测Notch3-siRNA对小鼠淋巴细胞增殖的抑制作用;EMSA检测NF-κB活性。在淋巴细胞体外培养试验中,转染Notch3 siRNA可以明显降低小鼠淋巴细胞内Notch3的表达,下调Notch3可以显著抑制刀豆蛋白A(ConA)诱导的T细胞CD69的表达;同时下调Notch3对小鼠淋巴细胞的增殖有抑制作用;而且发现下调Notch3能明显抑制ConA诱导的NF-κB活化。实验结果提示,下调Notch3信号可通过抑制NF-κB活化对小鼠T淋巴细胞活化与增殖发挥抑制作用。     

结核杆菌抗原特异性激发人γδT细胞活化信号涉及Ras/Erk途径

目的探讨Ras/Erk通路是否参与结核杆菌低分子多肽抗原(Mtb-Ag)启动的γδT细胞活化信号转导途径.方法分离获取PBMC,用佛波醇酯(PMA)和离子霉素(IM),CD3mAb或Mtb-Ag刺激不同时间,或PBMC先经PD98059处理后再刺激培养;用荧光单抗染色后在流式细胞仪测定细胞CD69分子的表达;计数培养扩增10天的细胞数量.结果PBMC用PMA+IM刺激6小时,总T细胞和γδT细胞中的CD69表达均达高峰(均＞99%),用CD3mAb刺激24小时,均达高峰(均在55%左右),用Mtb-Ag刺激24小时,亦均达高峰,但总T细胞和γδT细胞中CD69分别为16.0%和75.2%;用PD98059预处理PBMC,可明显抑制Mtb-Ag激活的γδT细胞CD69表达和γδT细胞的增殖.结论Mtb-Ag可特异性激活γδT细胞,其活化信号转导需通过Ras/Erk通路.     

漆黄素对巨噬细胞和T淋巴细胞的免疫调节作用

目的探讨漆黄素(Fisetin,FIS)对小鼠巨噬细胞吞噬功能、NO的释放及对T淋巴细胞体外活化、增殖的影响。方法无菌制备小鼠巨噬细胞悬液及淋巴细胞悬液;荧光微球结合流式细胞术(FCM)分析FIS对巨噬细胞吞噬作用的影响;Griess试剂盒检测巨噬细胞NO的释放;双色荧光抗体染色结合FCM,检测CD3+T细胞CD69的表达水平;CFSE标记技术检测T细胞增殖的情况。结果 2.5μmol/L,5μmol/L,10μmol/LFIS均能明显抑制巨噬细胞的吞噬微球的能力;FIS能抑制LPS和IFN-γ刺激的巨噬细胞的NO的产生(P<0.05);FIS对ConA刺激的T细胞表达CD69有抑制作用,并能有效抑制ConA诱导的T细胞增殖(P<0.01),且均呈剂量依赖性。结论 FIS能显著抑制小鼠腹腔巨噬细胞的吞噬能力和分泌NO的能力,并能够抑制T细胞的活化和增殖,有望发展成为一种新的免疫抑制药物。     

CD69分子在人外周血佛波醇酯+离子霉素活化的γδT细胞表面表达的实验研究

目的 观察CD69分子在人外周血佛波醇酯(PMA)+离子霉素(IM)活化的γδT细胞表面的表达情况.方法 分离获取健康人外周血单个核细胞(PBMC),用PMA+ IM刺激,采用流式细胞术检测CD3+细胞和γδT细胞0、6、12、24、48和72 h CD69分子的动态表达情况.结果 PMA+ IM刺激PBMC 0、6、12、24、48和72 h,CD3+细胞CD69分子的表达分别为1.0％、99.5％、99.1％、98.0％、93.3％、92.1％;γδT细胞CD69分子的表达分别为0.91％、99.3％、98.6％、93.6％、88.7％、87.0％.结论 PMA+ IM的刺激后CD3+细胞和γδT细胞迅速活化,CD69的表达几乎为100％,6h达高峰,随后缓慢下降,两种细胞活化表达CD69基本一致.     

Protease‐activated receptor 2 signalling promotes dendritic cell antigen transport and T‐cell activation in vivo

Deficiency of protease-activated receptor-2 (PAR2) modulates inflammation in several models of inflammatory and autoimmune disease, although the underlying mechanism(s) are not understood. PAR2 is expressed on endothelial and immune cells, and is implicated in dendritic cell (DC) differentiation. We investigated in vivo the impact of PAR2 activation on DCs and T cells in PAR2 wild-type (WT) and knockout (KO) mice using a specific PAR2 agonist peptide (AP2). PAR2 activation significantly increased the frequency of mature CD11c^high DCs in draining lymph nodes 24 hr after AP2 administration. Furthermore, these DCs exhibited increased expression of major histocompatibility complex (MHC) class II and CD86. A significant increase in activated (CD44⁺ CD62⁻) CD4⁺ and CD8⁺ T-cell frequencies was also observed in draining lymph nodes 48 hr after AP2 injection. No detectable change in DC or T-cell activation profiles was observed in the spleen. The influence of PAR2 signalling on antigen transport to draining lymph nodes was assessed in the context of delayed-type hypersensitivity. PAR2 WT mice that were sensitized by skin-painting with fluorescein isothiocyanate (FITC) to induce delayed-type hypersensitivity possessed elevated proportion of FITC⁺ DCs in draining lymph nodes 24 hr after FITC painting when compared with PAR2 KO mice (0·95% versus 0·47% of total lymph node cells). Collectively, these results demonstrate that PAR2 signalling promotes DC trafficking to the lymph nodes and subsequent T-cell activation, and thus provides an explanation for the pro-inflammatory effect of PAR2 in animal models of inflammation.     

Regulation of human mesenchymal stem cell differentiation by TREM-2

Activation of the triggering receptor expressed on myeloid cells 2 (TREM-2) regulates myeloid cell function in vitro. However, the failure to detect TREM-2 protein expression in vivo has hampered studies on immunological and other physiological TREM-2 functions. This study demonstrates that TREM-2 is expressed by human mesenchymal stem cells (h-MSCs) and responds to the toll-like receptor (TLR) ligand lipopolysaccharide (LPS). Knockdown of TREM-2 in h-MSCs using a small interfering RNA (siRNA) reduced the expression levels of TLR2, TLR4, and TLR6, inhibited osteogenic, chondrogenic, and adipogenic differentiation under specific induction conditions, and enhanced LPS-evoked inflammatory cytokine production. Thus, activation of TREM-2 may restrain h-MSC immune activation and promote differentiation for tissue repair.     

T-cell surface molecules involved in the induction and expression of lymphokine-activated killing of autologous and allogeneic tumor targets

The cellular interactions and the surface molecules involved in the generation and the expression of lymphokine-activated killer-cell (LAK) activitiesin vitro in blood mononuclear cells (BMN) from cancer patients and healthy individuals against autologous and allogeneic tumors were studied. The depletion of a plastic-adherent population(s) from BMN at the initiation ofin vitro cultures in recombinant interleukin-2 (rIL-2) markedly interfered with the generation of LAK activities. Readdition of the same number of irradiated autologous plastic-adherent cells to the nonadherent population restored the generation of LAK. The requirement of the plasticadherent population(s) inin vitro induction of LAK activities was observed only in autologous situations. Furthermore, selective modulations of CD3 and CD2 receptors on BMN with the appropriate monoclonal antibodies (MAb) during the induction phases of LAK responses profoundly inhibited the generation of LAK. Thus, unhindered expressions of CD2 molecules and CD3 molecules were necessary for the maximum cytotoxic activation of non-antigen-driven effector cells in short-term cultures in rIL-2.     

锂增强小鼠LAK细胞活性及其体内抗瘤作用的研究

本文观察了锂体外对小鼠ＬＡＫ细胞的作用及其体内抗瘤作用，以期找到一种能增强ＬＡＫ细胞活性，降低ＩＬ－２毒副作用的免疫调变剂。在ＩＬ－２诱导ＬＡＫ的同时加入ＬｉＣｌ，则能增强ＬＡＫ细胞的活性。以Ｃ５７ＢＬ／６小鼠接种Ｂ１６黑色素瘤细胞为荷瘤小鼠模型，比较了ＩＬ－２／ＬＡＫ，单独锂及ＩＬ－２／ＬＡＫ合并锂三组的抗瘤作用，结果表明，单独锂及ＩＬ－２／ＬＡＫ组与对照组相比均有抗瘤作用，但ＩＬ－２／ＬＡＫ和锂一起作用，其抗瘤作用最强，表现为能抑制肿瘤的生长及延长荷瘤小鼠的存活时间。因此，锂可望作为一种新型的免疫调节剂用于免疫性疾病及肿瘤的治疗。     

血小板活化因子对大鼠卵巢颗粒细胞E2分泌的影响及机制

目的：观察血小板活化因子（PAF）对体外培养的大鼠卵巢颗粒细胞E2分泌的影响,初步探讨其可能的作用机制。方法：原代细胞培养和放免技术。结果：PAF、FSH、forscolin、PMA都能明显刺激大鼠卵巢颗粒细胞E2的分泌（P〈0.05）,PAF对FSH、forscolin、PMA诱导的大鼠卵巢颗粒细胞E2分泌均有明显的增强作用（P〈0.05）。结论：PAF能明显促进体外培养的大鼠卵巢颗粒细胞E2的分泌,并且受FSH及PKA、PKC信号转导通路的调节。     

Bone morphogenetic protein is required for fibroblast growth factor 2-dependent later-stage osteoblastic differentiation in cranial suture cells

Background: Understanding the pathophysiological process of calvarial bones development is important for the treatments on relative diseases such as craniosynostosis. While, the role of fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) and how they interacted in osteoblast differentiation remain unclear. Methods: we digested bone fragments around the coronal and sagittal sutures from newborn rats to harvest suture cells. Markers expression at different osteoblast differentiation stage was analyzed by increasing FGF2 concentration and BMP2 blocking in these cells. Results: BMP2 expression could be stimulated by FGF2 in a dose and time dependent manner. FGF2 stimulation may decrease early marker of osteoblast differentiation (collagen type-1, COL-1) and increase the expression of continuously-expressed or late markers (alkaline phosphatase, ALP; osteocalcin, OC and bone sialoprotein, BSP) to accelerate mineralization. Inhibition of BMP2 signaling by Noggin weakens the effect of FGF2 on induction of later-stage osteoblastic differentiation of cranial suture cells. Conclusion: Our data suggest that BMP2 signaling is required for FGF2-dependent induction of later-stage of cranial suture cell osteoblastic differentiation.     

Encapsulation of mammalian cells into synthetic polymer membranes using least toxic solvents

Immunoisolation, that is, enclosure of cells within a semipermeable membrane to protect them from immunological rejection, may enable the transplantation of cells without use of immunosuppressive drugs. Therefore, in addition to naturally-occurring ionic polymers, several synthetic nonionic polymers which can form dense and strong membranes in water have been studied as materials for immunoisolation. However, such nonionic polymers are required to be soluble in organic solvents which are mostly cytotoxic. In this report we describe enclosure of insulin-releasing cells into water-insoluble poly(2-hydroxyethyl methacrylate) and poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) membranes using X-ray contrast medium as a solvent without use of any special apparatus. The contrast medium employed in our study is iopamidol aqueous solution. Insulin release was observed for 1 month when insulin-releasing cells were encapsulated into these membranes. The permeability of five solutes through the membranes prepared from the iopamidol aqueous solution was also studied to determine their potential immunoisolative efficacy.     

高温与阿霉素对兔VX-2细胞的协同作用

以体外长期培养的兔ＶＸ－２细胞为靶细胞,采用水浴加法进行体外细胞毒试验,（ＭＴＴ法）,观察了热疗,化疗及热化疗对兔ＶＸ－２细胞的生长抑制规律的影响,结果表明：（１）温热和阿纱对ＶＸ－２均有一定的抑制和杀伤作用两者一定方式的联合具有协同或相加作用。（２）热化疗加热温度以４２～４３℃为柱,加热时间以３０ｍｉｎ以上为佳。     

神经肽对肥大细胞内钙离子水平的影响

采用新型的钙荧光指示剂Ｆｕｒａ－Ⅱ检测了神经肽刺激后ＲＰＭＣ内ｃａ２＋的变化情况。在细胞外无钙离子存在下，用能诱导组胺释放的最适浓度的Ｐ物质（１０μｍｏｌ／Ｌ）、β－内啡肽（６μｍｏｌ／Ｌ）和强啡肽（２μｍｏｌ／Ｌ）刺激ＲＰＭＣ后，胞内Ｃａ２＋迅速上升，然后很快下降，该过程约需１５秒钟左右。这与组胺释放的时间基本吻合。推测上述三种神经肽触发胞内钙池释放Ｃａ２＋可能与组胺的释放有关。