rhG-CSF动员对供者CD4^＋T细胞免疫表型和功能影响

目的：研究重组人粒细胞集落刺激因子（rhG-CSF）动员对供者CD4＋T细胞表面分子淋巴细胞功能相关抗原-1（LFA-1）、细胞间黏附分子-1（ICAM-1）、L-选择素（LAM-1）和人整合素-4（VLA-4）的表达及其介导的CD4＋T细胞功能的影响,探讨外周血干细胞移植过程中CD4＋T细胞免疫耐受机制。方法：使用三色荧光标记检测动员前及动员后第5天供者外周血LFA-1、ICAM-1、LAM-1和VLA-4的表达率,ELISA方法检测动员前后CD4＋T细胞分泌IFN-γ和IL-4能力,免疫磁性分选法分离纯化CD4＋T细胞,检测动员前后CD4＋T细胞对基质细胞衍生因子-1α（SDF-1α）的迁移能力和对ICAM-1的黏附能力。结果：动员前后CD4＋T细胞LFA-1（CD11a）和VLA-4（CD49d）表达率差异无统计学意义（P〉0.01）,动员前后CD4＋T细胞LAM-1（CD62L）和ICAM-1（CD54）的表达率差异均有统计学意义,动员前显著高于动员后（P〈0.01）;动员前后CD4＋T淋巴细胞向SDF-1α的迁移率差异无统计学意义（P〉0.01）;动员后CD4＋T细胞对ICAM-1的黏附率降低（P〈0.01）;动员后IL-4和IFN-γ两个细胞因子在外周血血清的浓度均降低（P〈0.01）。结论：rhG-CSF动员不影响CD4＋T细胞LFA-1和VLA-4表达及CD4＋T细胞迁移,但影响CD4＋T细胞ICAM-1和LAM-1表达以及CD4＋T细胞通过LFA-1对ICAM-1的黏附能力影响,并可能影响CD4＋T细胞分泌细胞因子IL-4及IFN-γ的功能。     

Th1细胞因子(IFN-γ)在幽门螺杆菌致病中的作用

目的：评价Thl细胞因子IFN-γ在幽门螺杆菌(Hp)感染时对胃上皮细胞的作用。方法：胃上皮细胞经IFN-γ处理后,流式细胞术测定表面MHC-Ⅱ类分子的表达和Hp的黏附,ELISA法测定细胞因子对Hp致胃上皮细胞凋亡的影响。结果：IFN-γ可诱导胃上皮细胞表达MHCR类分子,进而增加Hp的黏附。IFN-γ本身即可诱导胃上皮细胞凋亡,并可促进Hp诱导的胃上皮凋亡。结论：Thl细胞因子IFN-γ参与并加剧了Hp感染所致的胃黏膜炎症。     

rhG-CSF 动员对供者CD4+ T 细胞免疫表型和功能影响

目的:研究重组人粒细胞集落刺激因子(rhG-CSF)动员对供者CD4+T细胞表面分子淋巴细胞功能相关抗原-1(LFA-1)、细胞间黏附分子-1(ICAM-1)、L-选择素(LAM-1)和人整合素-4(VLA-4)的表达及其介导的CD4+T细胞功能的影响,探讨外周血干细胞移植过程中CD4+T细胞免疫耐受机制。方法:使用三色荧光标记检测动员前及动员后第5天供者外周血LFA-1、ICAM-1、LAM-1和VLA-4的表达率,ELISA方法检测动员前后CD4+T细胞分泌IFN-γ和IL-4能力,免疫磁性分选法分离纯化CD4+T细胞,检测动员前后CD4+T细胞对基质细胞衍生因子-1α(SDF-1α)的迁移能力和对ICAM-1的黏附能力。结果:动员前后CD4+T细胞LFA-1(CD11a)和VLA-4(CD49d)表达率差异无统计学意义(P>0.01),动员前后CD4+T细胞LAM-1(CD62L)和ICAM-1(CD54)的表达率差异均有统计学意义,动员前显著高于动员后(P<0.01);动员前后CD4+T淋巴细胞向SDF-1α的迁移率差异无统计学意义(P>0.01);动员后CD4+T细胞对ICAM-1的黏附率降低(P<0.01);动员后IL-4和IFN-γ两个细胞因子在外周血血清的浓度均降低(P<0.01)。结论:rhG-CSF动员不影响CD4+T细胞LFA-1和VLA-4表达及CD4+T细胞迁移,但影响CD4+T细胞ICAM-1和LAM-1表达以及CD4+T细胞通过LFA-1对ICAM-1的黏附能力影响,并可能影响CD4+T细胞分泌细胞因子IL-4及IFN-γ的功能。     

白细胞介素21对SHIV感染的恒河猴外周血CD8~＋ T细胞分泌γ干扰素的影响

目的研究白细胞介素21（interleukin 21,IL-21）对SHIV感染CD8＋T细胞分泌干扰素γ（interferon-γ,IFN-γ）的影响。方法从SHIV/恒河猴模型外周血中分选出CD8＋T细胞,加入IL-21诱导培养,应用ELISA方法检测细胞培养上清液中IFN-γ浓度,RT-PCR方法检测细胞中IFN-γmRNA的表达水平,流式细胞术检测分泌IFN-γ的CD8＋T细胞所占的百分比。结果 10 ng/mL IL-21明显促进CD8＋T细胞分泌IFN-γ（P〈0.05）,IFN-γmRNA的表达明显升高,4 h为刺激CD8＋T细胞胞内IFN-γ合成的最佳时间。结论 IL-21对CD8＋T细胞分泌IFN-γ有促进作用。     

腺苷酸活化蛋白激酶活化对单核细胞-内皮细胞黏附的影响及其机制北大核心CSCD

本研究旨在探讨腺苷酸活化蛋白激酶（AMP-activated protein kinase,AMPK）活化对单核细胞与内皮细胞黏附的影响及其分子机制。用不同剂量的AMPK激动剂5-氨基咪唑-4-甲酰胺核糖核苷酸（AICAR,0~2 mmol/L）或AMPK抑制剂compound C（10 mmol/L）处理肿瘤坏死因子α（tumor necrosis factorα,TNFα,10 ng/m L）诱导的人主动脉内皮细胞（human aortic endothelial cells,HAECs）,用TNFα诱导过表达活性型或显性抑制型AMPK蛋白的HAECs。用荧光染色法观察AMPK对荧光标记的单核THP-1细胞与HAECs黏附的影响。用荧光定量PCR检测血管细胞黏附分子1（vascular cell adhesion molecule-1,VCAM-1）和细胞间黏附分子1（intercellular cell adhesion molecule-1,ICAM-1）m RNA表达水平,用ELISA法检测二者的蛋白分泌量;用Western blot检测核因子-kappa B（nuclear factor-kappa B,NF-κB）p65的211位点赖氨酸乙酰化水平,用ELISA法检测NF-κB p65DNA结合活性,并用试剂盒检测p300乙酰转移酶活性。通过小干扰RNA抑制HAECs组蛋白乙酰转移酶p300蛋白表达后,检测TNFα对NF-κB p65 DNA结合活性、黏附分子ICAM-1、VCAM-1的表达及单核细胞黏附率的影响。结果显示,AICAR显著抑制TNFα诱导的单核细胞与HAECs的黏附,在HAECs中下调TNFα诱导的ICAM-1、VCAM-1的m RNA水平上调和蛋白分泌。AICAR的效应可以被AMPK抑制剂compound C完全阻断。转染活性型AMPKα显著抑制TNFα诱导的ICAM-1、VCAM-1m RNA表达和分泌,以及单核细胞-内皮细胞黏附,而转染显性抑制型AMPKα则无明显影响。RNAi干预抑制p300活性显著抑制TNFα诱导的黏附分子表达和单核-内皮细胞黏附。AMPK激活可抑制TNFα诱导的p300乙酰转移酶活性,抑制NF-κB p65的211位赖氨酸的乙酰化,降低NF-κB p65 DNA结合活性。以上结果提示,AMPK激活抑制单核细胞-内皮细胞黏附,作用机制可能与其降低p300酶活性,下调NF-κB p65转录活性密切相关。     

TNF-α在PMA和IFN-γ诱导U 937细胞生长和分化过程中的作用

本文报道了佛波酯(PMA)和γ-干扰素(IFN-γ)对U937细胞生长和分化的调控作用及其机制。PMA和IFN-γ能以剂量依赖的方式诱导U937细胞向成熟单核/巨噬细胞样细胞分化,同时抑制其细胞的生长。实验发现PMA和IFN-γ可诱导U937细胞表达TNF-α特异性mRNA和蛋白质。U937细胞培养中加入特异性抗TNF-α抗体可以抑制PMA和IFN-γ诱导U937细胞的分化和生长。这说明内源性的TNF-α在介导PMA和IFN-γ上述生物效应过程中起一定作用。TNF-α对U937细胞的这种调节作用与其胞毒作用机制不同     

球形孢子丝菌刺激小鼠树突状细胞表达前炎症细胞因子的研究

目的本研究旨在观察分离于新疆的球形孢子丝菌临床株刺激小鼠树突状细胞(Dendritic cells,DCs)后不同炎症因子分泌表达的特征,初步预测这些炎症因子的功能。方法菌株来源于淋巴管型孢子丝菌病患者。将该菌配置成不同浓度的菌悬液(1×10~4个/mL～1×10~7个/mL),刺激小鼠DC(细胞悬浮液浓度1×10~6细胞/mL),分别收集6 h、24 h、48 h、72 h时细胞培养上清液,采用酶免法检测IL-1β、IL-6、IL-4、TNF-α、IFN-γ的含量表达。结果以最低浓度菌液(1×10~4个/mL)刺激DC,被刺激后的DC分泌了IL-1β、IL-6和TNF-α,不同时间点分泌量分别为:IL-1β(6 h:21.26±3.03;24 h:24.04±4.25;48 h:24.90±4.31;72 h:27.29±6.09)、IL-6(6 h:44.38±3.73;24 h:101.72±12.28;48 h:133.10±8.67;72 h:180.38±13.84)、TNF-α(6 h:860.36±20.64;24 h:356.03±11.46;48 h:457.43±17.39;72 h:1454.53±19.46),但是IFN-γ和IL-4未见分泌表达。IL-1β和IL-6分泌水平有随时间和剂量依赖而逐渐增高,但是TNF-α释放量呈现不规律表达。结论 DC参与了球形孢子丝菌感染的天然免疫应答,分泌的关键炎症因子是IL-1β、IL-6、和TNF-α,表达量为TNF-α IL-6 IL-1β。其中IL-1β和IL-6分泌表达量具有时间和剂量依赖性。     

肾康灵调控系膜细胞炎性反应因子的机制研究

目的：观察氧化低密度脂蛋白（Oxidized Low-Density Lipoprotein,ox-LDL）对系膜细胞（Mesangial Cells,MCs）分泌炎性反应递质功能的影响,并从细胞分子生物学水平阐明肾康灵的作用机制。方法：采用肾康灵干预增殖的系膜细胞,并利用分子生物学技术检测CXCL16、CD36、IFN-γ、IL6、TNF-α含量或基因水平。结果：利用ox-LDL诱导大鼠系膜细胞增殖并加入CXCR6受体后,CXCL16、CD36、IFN-γ、IL6、TNF-α的含量或基因水平显著升高,肾康灵呈浓度依赖性降低其表达水平。结论：ox-LDL诱导系膜细胞增殖时通过CXCR6介导,可促进CXCL16、CD36、IFN-γ、IL6、TNF-α等炎性反应递质的释放,中药肾康灵可通过抑制炎性反应递质的释放,保护系膜细胞的功能。     

卡介苗作用大鼠膀胱肿瘤细胞／正常移行上皮细胞后TNF—α．、IL-10、IFN-γ的检测及意义

目的：观察卡介苗（BCG）单独作用膀胱肿瘤细胞、正常膀胱移行上皮细胞及其代谢产物作用上述细胞后细胞生长情况及各自细胞培养液上清液中细胞因子（TNF-α．、IL-10、IFN-γ）浓度的变化,探讨其在卡介苗治疗膀胱肿瘤中可能的作用机制。方法：构建大鼠膀胱肿瘤模型,并原代培养大鼠膀胱肿瘤细胞及正常膀胱移行上皮细胞。分别用BCG,普通培养液和细胞培养的代谢产物作用上述细胞。酶联接免疫吸附剂测定法（ELISA法）检测各组细胞上清液中肿瘤坏死因子-α（TNF-α）、白细胞介素-10（IL-10）、干扰素-γ（IFN-γ）的浓度。结果：ELISA法检测各组细胞上清液中TNF-α、IL-10的浓度改变有显著差异,而IFN-γ的浓度无显著差异。结论：BCG可以直接刺激肿瘤细胞自身分泌细胞因子（TNF-α、IL-10）参与调节抑制肿瘤细胞的生长。     

Expression of semaphorin 3A in the rat corneal epithelium during wound healing

The neural guidance protein semaphorin 3A (Sema3A) is expressed in corneal epithelial cells of the adult rat. We have now further investigated the localization of Sema3A in the normal rat corneal epithelium as well as changes in its expression pattern during wound healing after central corneal epithelial debridement. The expression pattern of Sema3A was compared with that of the tight-junction protein zonula occludens-1 (ZO-1), the gap-junction protein connexin43 (Cx43), or the cell proliferation marker Ki67. Immunofluorescence analysis revealed that Sema3A was present predominantly in the membrane of basal and wing cells of the intact corneal epithelium. The expression of Sema3A at the basal side of basal cells was increased in the peripheral epithelium compared with that in the central region. Sema3A was detected in all layers at the leading edge of the migrating corneal epithelium at 6 h after central epithelial debridement. The expression of Sema3A was markedly up-regulated in the basal and lateral membranes of columnar basal cells apparent in the thickened, newly healed epithelium at 1 day after debridement, but it had largely returned to the normal pattern at 3 days after debridement. The expression of ZO-1 was restricted to superficial epithelial cells and remained mostly unchanged during the wound healing process. The expression of Cx43 in basal cells was down-regulated at the leading edge of the migrating epithelium but was stable in the remaining portion of the epithelium. Ki67 was not detected in basal cells of the central epithelium at 1 day after epithelial debridement, when Sema3A was prominently expressed. Immunoblot analysis showed that the abundance of Sema3A in the central cornea was increased 1 day after epithelial debridement, whereas that of ZO-1 or Cx43 remained largely unchanged. This increase in Sema3A expression was accompanied by up-regulation of the Sema3A coreceptor neuropilin-1. Our observations have thus shown that the expression of Sema3A is increased markedly in basal cells of the newly healed corneal epithelium, and that this up-regulation of Sema3A is not associated with cell proliferation. They further suggest that Sema3A might play a role in the regulation of corneal epithelial wound healing.     

益生菌对胆汁淤积性黄疸大鼠小肠上皮细胞紧密连接蛋白的影响

目的探讨双歧三联活菌(培菲康)对胆汁淤积性大鼠小肠上皮细胞紧密连接蛋白ZO-1(zonula oc-cludens-1)和Occludin表达的调控机制。方法雄性3周龄SD大鼠随机分为对照组、模型组和培菲康组,模型组和培菲康组均给予α-异硫氰酸萘酯(ANIT)50 mg/kg一次性灌胃,建立急性肝内胆汁淤积动物模型,培菲康组于造模前4 d开始给予培菲康4.2×107个活菌数/(kg.d)灌胃大鼠。分别于造模后24、48和72 h三个时间点处死大鼠,取末端回肠黏膜组织,采用免疫组化和Western blots免疫印迹法检测紧密连接蛋白ZO-1、闭锁蛋白(Occludin)的分布和表达,并利用图像分析系统对Western blots图像结果进行定量分析。结果ZO-1和Occludin蛋白主要沿大鼠小肠黏膜上皮细胞膜的顶端呈线状分布,模型组大鼠24 h时ZO-1和Occludin的阳性染色较对照组减少,48 h减少最为明显,72 h阳性染色有所恢复,而培菲康组大鼠各时间点ZO-1和Occludin的阳性染色和模型组相比均明显增多。Western blots结果与免疫组织化学结果相一致,模型组24 h已经开始下降(ZO-1 0.1294±0.0481)、(Oc-cludin 0.1950±0.0441),48 h达到最低(ZO-1 0.0395±0.0095)、(Occludin 0.0137±0.0092),72 h开始恢复(ZO-10.2024±0.0498)、(Occludin 0.1494±0.0355),各时间点与对照组(ZO-1 0.2887±0.0237)、(Occludin 0.4266±0.0670)相比差异有统计学意义(P0.01);而培菲康组各时间点蛋白表达分别为24 h(ZO-1 0.2110±0.0367)、(Occludin 0.3056±0.0572),48 h(ZO-1 0.1173±0.0423)、(Occludin 0.0521±0.0123),72 h(ZO-1 0.2601±0.0191)、(Occludin 0.2050±0.0721),与模型组相应时间点数据相比差异有统计学意义(P0.05)。结论双歧三联活菌能够影响胆汁淤积性大鼠小肠黏膜上皮紧密连接蛋白的分布和表达,可以恢复肠黏膜上皮屏障的完整性。     

转化生长因子-β1对大鼠骨髓间充质干细胞增殖及Slug表达的影响

目的检测转化生长因子-β1(transforming growth factor-β1,TGF-β1)对体外培养大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMMSCs)增殖及Slug表达的影响。方法采用密度梯度离心结合贴壁法分离、培养大鼠BMMSCs,用免疫组织化学方法对培养第3代的细胞进行鉴定。用MTT法检测不同浓度TGF-β1对细胞增殖的影响;免疫荧光和免疫印迹法检测TGF-β1处理前后Slug的表达情况。结果密度梯度离心结合贴壁法能有效分离、纯化大鼠BMMSCs,免疫组织化学法检测显示CD29、CD44表达阳性,而CD34、CD45表达阴性;低浓度TGF-β1对BMMSCs的增殖有促进作用,高浓度却抑制BMMSCs的增殖。TGF-β1处理24 h,Slug蛋白表达明显增强。结论一定浓度的TGF-β1可以促进BMMSCs的增殖,而且引起Slug蛋白增加。     

A comparative evaluation of corneal epithelial cell cultures for assessing ocular permeability

The purpose of this study was to evaluate the potential value of different epithelial cell culture systems as in vitro models for studying corneal permeability. Transformed human corneal epithelial (HCE-T) cells and Statens Serum Institut rabbit corneal (SIRC) cells were cultured on permeable filters. SkinEthic human corneal epithelium (S-HCE) and Clonetics human corneal epithelium (C-HCE) were received as ready-to-use systems. Excised rabbit corneas (ERCs) and human corneas (EHCs) were mounted in Ussing chambers, and used as references. Barrier properties were assessed by measuring transepithelial electrical resistance, and by determining the apparent permeability of markers with different physico-chemical properties, namely, fluorescein, sodium salt; propranolol hydrochloride; moxaverine hydrochloride; timolol hydrogenmaleate; and rhodamine 123. SIRC cells and the S-HCE failed to develop epithelial barrier properties, and hence were unable to distinguish between the permeation markers. Barrier function and the power to differentiate compound permeabilities were evident with HCE-T cells, and were even more pronounced in the case of C-HCE, corresponding very well with data from ERCs and EHCs. A net secretion of rhodamine 123 was not observed with any of the models, suggesting that P-glycoprotein or similar efflux systems have no significant effects on corneal permeability. Currently available corneal epithelial cell culture systems show differences in epithelial barrier function. Systems lacking functional cell-cell contacts are of limited value for assessing corneal permeability, and should be critically evaluated for other purposes.     

The immunoreactivity of a chimeric multi-epitope DNA vaccine against IBV in chickens

Lumican is a major proteoglycans of the human cornea. Lumican knock-out mice have been shown to lose corneal transparency and to display delayed wound healing. The purpose of this study was to define the role of lumican in corneal epithelial cell migration. Over-expression of lumican in human corneal epithelial (HCE-T) cells increased both cell migration and proliferation, and increased levels of integrins α2 and β1. ERK 1/2 was also activated in lumican over-expressed cells. When we treated HCE-T cells with the ERK-specific inhibitor U0126, cell migration and the expression of integrin β1 were completely blocked. These data provide evidence that lumican stimulates cell migration in the corneal epithelium by activating ERK 1/2, and point to a novel signaling pathway implicated in corneal epithelial cell migration.     

Human Bone Marrow Mesenchymal Stem Cells Induce Collagen Production and Tongue Cancer Invasion

Tumor microenvironment (TME) is an active player in carcinogenesis and changes in its composition modify cancer growth. Carcinoma-associated fibroblasts, bone marrow-derived multipotent mesenchymal stem cells (BMMSCs), and inflammatory cells can all affect the composition of TME leading to changes in proliferation, invasion and metastasis formation of carcinoma cells. In this study, we confirmed an interaction between BMMSCs and oral tongue squamous cell carcinoma (OTSCC) cells by analyzing the invasion progression and gene expression pattern. In a 3-dimensional myoma organotypic invasion model the presence of BMMSCs inhibited the proliferation but increased the invasion of OTSCC cells. Furthermore, the signals originating from OTSCC cells up-regulated the expression of inflammatory chemokines by BMMSCs, whereas BMMSC products induced the expression of known invasion linked molecules by carcinoma cells. Particularly, after the cell-cell interactions, the chemokine CCL5 was abundantly secreted from BMMSCs and a function blocking antibody against CCL5 inhibited BMMSC enhanced cancer invasion area. However, CCL5 blocking antibody did not inhibit the depth of invasion. Additionally, after exposure to BMMSCs, the expression of type I collagen mRNA in OTSCC cells was markedly up-regulated. Interestingly, also high expression of type I collagen N-terminal propeptide (PINP) in vivo correlated with the cancer-specific mortality of OTSCC patients, whereas there was no association between cancer tissue CCL5 levels and the clinical parameters. In conclusion, our results suggest that the interaction between BMMSC and carcinoma cells induce cytokine and matrix molecule expression, of which high level of type I collagen production correlates with the prognosis of OTSCC patients.     

Deletion of JAM-A causes morphological defects in the corneal epithelium

Junctional adhesion molecule-A (JAM-A, JAM-1, F11R) is an Ig domain containing transmembrane protein that has been proposed to function in diverse processes including platelet activation and adhesion, leukocyte transmigration, angiogenesis, epithelial cell shape and endothelial cell migration although its function in vivo is less well established. In the mouse eye, JAM-A protein expression is first detected at 12.5 dpc in the blood vessels of the tunica vasculosa, while it is first detected in both the corneal epithelium and lens between 13.5 and 14.5 dpc. In the corneal epithelium, JAM-A levels remain appreciable throughout life, while JAM-A immunostaining becomes stronger in the lens as the animals age. Both the cornea and lens of mice lacking an intact JAM-A gene are transparent until at least a year of age, although the cells of the JAM-A null corneal epithelium are irregularly shaped. In wild-type mice, JAM-A protein is found at the leading edge of repairing corneal epithelial wounds, however, corneal epithelial wound repair was qualitatively normal in JAM-A null animals. In summary, JAM-A is expressed in the corneal epithelium where it appears to regulate cell shape.     

CD200 positive human mesenchymal stem cells suppress TNF-alpha secretion from CD200 receptor positive macrophage-like cells

Human mesenchymal stem cells (hMSCs) display immunosuppressive properties in vitro and the potential has also been transferred successfully to clinical trials for treatment of autoimmune diseases. OX-2 (CD200), a member of the immunoglobulin superfamily, is widely expressed in several tissues and has recently been found from hMSCs. The CD200 receptor (CD200R) occurs only in myeloid-lineage cells. The CD200-CD200R is involved in down-regulation of several immune cells, especially macrophages. The present study on 20 hMSC lines shows that the CD200 expression pattern varied from high (CD200Hi) to medium (CD200Me) and low (CD200Lo) in bone marrow-derived mesenchymal stem cell (BMMSC) lines, whereas umbilical cord blood derived mesenchymal stem cells (UCBMSCs) were constantly negative for CD200. The role of the CD200-CD200R axis in BMMSCs mediated immunosuppression was studied using THP-1 human macrophages. Interestingly, hMSCs showed greater inhibition of TNF-α secretion in co-cultures with IFN-γ primed THP-1 macrophages when compared to LPS activated cells. The ability of CD200Hi BMMSCs to suppress TNF-α secretion from IFN-γ stimulated THP-1 macrophages was significantly greater when compared to CD200Lo whereas UCBMSCs did not significantly reduce TNF-α secretion. The interference of CD200 binding to the CD200R by anti-CD200 antibody weakened the capability of BMMSCs to inhibit TNF-α secretion from IFN-γ activated THP-1 macrophages. This study clearly demonstrated that the efficiency of BMMSCs to suppress TNF-α secretion of THP-1 macrophages was dependent on the type of stimulus. Moreover, the CD200-CD200r axis could have a previously unidentified role in the BMMSC mediated immunosuppression.