氧化低密度脂蛋白对外周血内皮祖细胞数量和功能的影响

目的观察氧化低密度脂蛋白(Ox-LDL)对外周血内皮祖细胞(endothelial progenitor cell,EPC)数量和功能的影响.方法密度梯度离心法获取外周血单个核细胞,培养7天后,收集贴壁细胞并加入不同浓度Ox-LDL(分别为25μg/ml,50μg/ml,100μg/ml,200μg/ml)和LDL(100μg/ml)培养一定的时间(6 h,12 h,24h以及48h).激光共聚焦显微镜和流式细胞仪鉴定EPC,分别观察EPC的增殖、迁移、黏附和体外血管生成能力.结果Ox-LDL显著减少外周血EPC数量,200μg/ml Ox-LDL作用24h对EPC数量的影响最为显著(较对照组减少了近70%,P＜0.001).另外,100μg/ml Ox-LDL呈时间依赖关系减少EPC数量,于24h达到高峰(P＜0.01).Ox-LDL也显著损害了外周血EPC的黏附、迁移、增殖和体外血管生成能力.结论Ox-LDL可减少EPC数量并损害EPC功能.     

辛伐他汀对兔外周血内皮祖细胞增殖、迁移、黏附能力的影响

目的 观察辛伐他汀对兔外周血内皮祖细胞(EPC)增殖、迁移、黏附能力的影响.方法 采用密度梯度离心法从兔外周血获取单个核细胞,将其接种在纤维连接蛋白包被培养板,培养7 d后,收集贴壁细胞,加入不同浓度辛伐他汀(分别为0.3,0.8,2,5 mmol/L)培养一定的时间(6,12,24,48 h).通过激光共聚焦显微镜鉴定EPC,并在倒置荧光显微镜下计数.然后分别采用MTT比色法、改良的Boyden小室、黏附能力测定实验和体外血管生成试剂盒观察EPC的增殖能力、迁移能力、黏附能力和体外血管生成能力.结果 辛伐他汀显著增加兔外周血EPC数量,并且EPC数量随辛伐他汀浓度增加及作用时间延长而增加,4 mmol/L浓度辛伐他汀作用24 h对EPC数量的影响最为显著(P<0.01).辛伐他汀也显著改善了兔外周血EPC的黏附能力、迁移能力、增殖能力和体外血管生成能力.结论 辛伐他汀可增加EPC的数量且改善EPC的功能.     

黄芪甲苷对人外周血内皮祖细胞体外血管生成能力的影响

目的观察黄芪甲苷对体外培养的人外周血内皮祖细胞(EPCs)数量及增殖、迁移、黏附功能、血管生成能力的影响。方法密度梯度离心法获取人外周血单个核细胞,FITC标记荆豆凝集素Ⅰ(FITC-UEA-I)、DiI标记的乙酰化低密度脂蛋白(DiI-acLDL)荧光双染鉴定,流式细胞仪检测细胞表面标志。单个核细胞培养4d后进行实验分组,分为对照组和黄芪甲苷干预组。干预组加入不同浓度的黄芪甲苷(分别为10μg/mL、20μg/mL、40μg/mL)培养72h。分别采用四氮唑溴盐比色法、改良的Boyden小室和黏附能力测定来观察EPCs的增殖、迁移和黏附能力;体外血管生成试剂盒来观察EPCs体外血管生成能力。结果与正常对照组比较,黄芪甲苷组EPCs数量增加,EPCs的黏附、迁移、增殖和体外血管形成能力增强,且黄芪甲苷20μg/mL时作用最显著。结论黄芪甲苷可增加体外培养的EPCs数量,改善EPCs增殖、迁移、黏附和体外血管生成能力等生物学功能。     

通心络体外促进人外周血内皮祖细胞的增殖、迁移、黏附的研究

目的:研究中药通心络对体外培养的人外周血内皮祖细胞(EPCs)增殖、迁移、黏附的影响. 方法:采用密度梯度离心法分离培养人外周血单个核细胞,经FITC-UEA-I和Dil-acLDL双染色鉴定为正在分化的EPCs.进一步采用流式细胞仪检测其表面标志CD34、CD133,采用MTT比色法、transwell小室、细胞计数法分别观察50、100、200、500、750和1000μg/ml通心络超微粉溶液和500μg/ml通心络超微粉溶液作用0、6、12、24和36 h后,对EPCs增殖、迁移、黏附的影响. 结果:不同水平的通心络均改善了EPCs的增殖、迁移、黏附的功能,且在500μg/ml时作用最为显著.采用500μg/ml的通心络进行时效作用的研究显示,通心络呈时间依赖性地增强EPCs增殖、迁移、黏附能力,在36 h达到高峰(P＜0.01). 结论:通心络能显著改善外周血内皮祖细胞增殖、迁移、黏附能力,这可能是通心络防治心脑血管疾病的新机制.     

可溶性环氧化物水解酶抑制剂通过过氧化体增殖物激活型受体γ调节内皮祖细胞功能

目的研究可溶性环氧化物水解酶抑制剂t-AUCB对小鼠来源内皮祖细胞(EPC)功能的调节及相关机制。方法密度梯度离心法分离培养小鼠骨髓来源EPC,不同浓度t-AUCB及过氧化体增殖物激活型受体γ(PPARγ)阻断剂GW9662预干预EPC,检测上述EPC体外增殖、黏附、迁移、血管生成情况。结果在1~100μmol/L范围内,t-AUCB呈浓度依赖性增强EPC体外增殖、黏附、迁移、血管生成能力,而5μmol/L PPARγ阻断剂GW9662可抑制EPC上述功能。结论可溶性环氧化物水解酶抑制剂t-AUCB参与EPC增殖、黏附、迁移、血管生成等功能的调控,其机制可能与激活PPARγ通路有关。     

肿瘤坏死因子-α对内皮祖细胞功能及其一氧化氮合酶含量的影响

目的观察肿瘤坏死因子-α(TNF-α)对外周血内皮祖细胞(EPC)功能及一氧化氮合酶含量的影响。方法采用密度梯度离心法从外周血获得单个核细胞,将其接种在人纤维连接蛋白包被的培养板上,培养7d后收集贴壁细胞,加入不同浓度TNF-α(分别为0,10,20,50和100mg/L)分别培养0,6,12,24和48h。激光共聚焦显微镜鉴定异硫氰酸荧光黄标记荆豆凝集素Ⅰ和Dil标记的乙酰化低密度脂蛋白双染色阳性的细胞为正在分化的EPC;将其在倒置荧光显微镜下计数,然后分别采用MTT比色法、改良的Boyden小室、黏附能力测定实验和体外血管生成试剂盒来观察EPC的增殖、迁移、黏附和体外血管形成能力。免疫印迹杂交法(Westernblot)半定量测定诱导型和内皮型一氧化氮合酶(iNOS和eNOS)含量。结果TNF-α降低外周血EPC增殖、迁移、黏附、体外血管形成能力、iNOS和eNOS含量,并且EPC增殖、迁移、黏附、体外血管形成能力、iNOS和eNOS含量随TNF-α浓度与作用时间增加而降低。结论TNF-α降低EPC的增殖、迁移、黏附和体外血管形成能力,还减少EPC中iNOS和eNOS含量。     

不同剂量阿托伐他汀钙对冠心病患者内皮祖细胞水平的影响

目的通过人体药物临床试验,观察不同剂量阿托伐他汀钙对冠心病患者外周血内皮祖细胞(EPC)增殖、迁移、黏附及血管修复能力的影响。方法选择冠心病患者40例,根据服用阿托伐他汀钙的剂量不同,分为小剂量组(20mg)20例及大剂量组(40mg)20例。所有入选患者于服药前服药5、15、30、60、90和120d分别抽取外周血获取外周血单个核细胞,体外培养后进行细胞染色与鉴定、MTT比色法检测细胞增殖、检测EPC的迁移及黏附能力,流式细胞仪分析EPC标记表面CD34、CD133、人血管内皮细胞生长因子受体2(VEGFR-2)的表达。结果EPC的迁移能力在30d时最强,30d后大剂量组迁移能力的下降趋势显著高于小剂量组(P<0.05,P<0.01);EPC增殖、黏附和CD34的表达在60d时达高峰;CD133在30d时最低,而此后大剂量组成熟EPC的下降率显著高于小剂量组;VEGFR-2的细胞表达在60d后呈持续下降趋势,而小剂量组VEGFR-2明显高于大剂量组(P<0.05,P<0.01)。结论长期使用阿托伐他汀钙并不能有效促进EPC的活化及新生血管的生成。20mg的阿托伐他汀钙对血管内膜的修复更优于40mg。     

流体切应力对衰老内皮祖细胞血管内皮修复能力的影响及机制研究

目的探讨体外流体切应力对老年志愿者内皮祖细胞(EPC)功能活性的影响及可能的分子机制。方法抽取老年志愿者外周血20ml,采用密度梯度离心法分离提取EPC。在体外采用生理范围(0.15mN/cm2)流体切应力处理培养后的老年志愿者EPC 24h,观察EPC体外的迁移、黏附能力的变化。通过建立裸鼠颈动脉拉脱损伤模型,观察流体切应力对老年志愿者EPC裸鼠颈动脉内皮损伤修复能力的影响。实时RT-PCR和Western blot检测EPC表面相关基因和蛋白的表达情况。结果体外切应力干预明显提高老年志愿者EPC体外的迁移、黏附能力,0.15mN/cm2体外切应力干预24h能提高EPC修复损伤血管内皮的能力[干预前:(33.1±5.9)%;干预后:(49.1±7.1)%,P0.01]。基因和蛋白水平检测显示,EPC表面的趋化生长因子受体4(CXCR4)信号通路明显上调(P0.01)。结论流体切应力干预显著改善老年志愿者EPC血管内皮损伤修复能力,其机制可能与EPC表面的CXCR4信号通路密切相关。     

雷帕霉素对内皮祖细胞增殖、黏附及迁移能力影响的实验研究

目的药物洗脱支架可抑制支架处血管内膜再内皮化,但对骨髓源性内皮修复作用的影响尚不清楚,为此观察了雷帕霉素对内皮祖细胞(endothelial progenitor cells,EPC)增殖、黏附及迁移的影响。方法采用密度梯度离心法从大鼠骨髓获取单个核细胞,将其接种在纤维连接素包被培养板,加入不同浓度雷帕霉素(0.01～100ng/ml),培养12天后,激光共聚焦显微镜鉴定FITC-UEA-Ⅰ和DiI-acLDL双染阳性细胞为正在分化的EPC,并在倒置荧光显微镜下计数。收集培养8天贴壁细胞,分别加入不同浓度雷帕霉素(0.1～200ng/ml)培养12～96h。然后分别采用MTT比色法、改良的Boyden小室和黏附能力测定实验观察EPC的增殖、迁移和黏附能力。结果雷帕霉素显著抑制骨髓单个核细胞分化为EPC,0.1ng/ml浓度雷帕霉素作用12天,EPC减少了(59.3±5.8)%(P<0.01)。雷帕霉素也显著抑制EPC增殖,其抑制作用随雷帕霉素浓度增加及作用时间延长而增加,1ng/ml浓度雷帕霉素作用24h使EPC数量减少(41.5±2.2)%(P<0.01)。雷帕霉素也显著抑制EPC的黏附和迁移能力。结论雷帕霉素可抑制骨髓单个核细胞分化为EPC,并抑制EPC的增殖及迁移能力。     

姜黄素对内皮祖细胞功能及内皮型一氧化氮合酶的影响

目的观察姜黄素对体外培养的人外周血内皮祖细胞（EPCs）数量及功能的影响。方法采用贴壁选择法培养人外周EPCs,培养6 d后,收集贴壁细胞并加入姜黄素（0、5、10、15μmol/L）培养一定时间（6、12、24和48 h）。荧光显微镜鉴定EPCs,分别观察EPCs的体外增殖、黏附、迁移、体外血管生成能力。Griess法检测NO分泌量,并RT-PCR半定量测定eNOS基因表达。结果姜黄素增加外周血EPCs的数量,改善EPCs的体外增殖、黏附、迁移、体外血管生成能力（P<0.01）。并上调eNOS基因表达,促进EPCs来源的NO的释放（P<0.01）。结论姜黄素增加体外EPCs数量及改善其功能,上调eNOS基因表达并增加EPCs来源的NO分泌。     

巴曲酶对体外培养下内皮祖细胞数量及功能的影响

目的探讨巴曲酶(DF-521)对内皮祖细胞(endothelial progenitor cells,EPCs)数量和功能的影响。方法采用密度梯度离心法从外周血获取单个核细胞,将其接种在细胞培养板中,分为对照组、低剂量干预组(DF-5210.05BU/ml)、中剂量干预组(DF-5210.1BU/ml)和高剂量干预组(DF-5210.2BU/ml)。激光共聚焦显微镜鉴定"FITC标记的荆豆凝集素"和"DiI标记的乙酰化低密度脂蛋白"双染色阳性细胞为正在分化的EPCs,流式细胞仪检测其表面标记并进一步鉴定EPCs。MTT比色法、改良的Boyden小室及黏附能力测定EPCs的增殖、迁移和黏附能力。结果与对照组比较,不同浓度DF-521干预组均随浓度增加明显提高EPCs的数量;中剂量干预组作用24h时对EPCs的数量影响最为显著,较对照组增加近1倍(P<0.05)。DF-521对于EPCs的增殖、迁移、黏附能力也有显著的改善(P<0.05)。结论DF-521可增加体外培养条件下EPCs的数量并改善其功能。     

U94 of human herpesvirus 6 inhibits in vitro angiogenesis and lymphangiogenesis

Human herpesvirus 6 (HHV-6) is a lymphotropic virus, but recent observations showed that also vascular endothelial cells (ECs) are susceptible to infection, both in vivo and in vitro. The observation that lymph nodes are a site of viral persistence suggests that lymphatic ECs (LECs) might be even more relevant for HHV-6 biology than vascular ECs. Here, we provide evidence that HHV-6 can infect LECs in vitro and establish a latent infection. Thus HHV-6 infection induces the loss of angiogenic properties both in LECs and in vascular ECs, as shown by the inability to form capillary-like structures and to seal wound scratches. The antiangiogenic effects observed in infected cells are associated to the expression of HHV-6 U94/rep, a latency-associated gene. In fact, transfection of U94/rep or addition of recombinant U94/REP protein to ECs inhibits the formation of in vitro capillary-like structures, reduces migration of ECs, and blocks angiogenesis, rendering rat aortic rings insensitive to VEGF-induced vasculogenetic activity. The ability of U94/rep to block different angiogenetic steps may lead to approaches in the potential control of the proliferation of blood and lymphatic vessels.     

Electron-microscopic aspects of Hodgkin's disease

Summary Lymph nodes from patients with Hodgkin's disease of the nodular sclerosis or mixed cellularity type were examined by electron microscopy to classify all the cells that occur in these types of lymphoma. Most of the cells showed morphological features that were the same, or nearly the same as those of cells of normal lymphoid tissue. These included typical interdigitating reticulum cells (IDC), histiocytic reticulum cells, so-called dark reticulum cells, and sinus macrophages. There were also small and medium-sized lymphocytes, immunoblasts, plasma cells, and plasma cell precursors resembling those seen in non-specific lymphadenitis. Germinal center cells, on the other hand, were present in negligible numbers.Special attention was paid to Hodgkin's (H) and Sternberg-Reed (SR) cells. This group of cells proved to be heterogeneous. The only common features were a large cell size, large nuclei, and a prominent nucleolus. Some of the H and SR cells resembled immunoblasts of normal lymphoid tissue. The cytoplasm of these cells contained numerous polyribosomes, and their heterochromatin was coarsely condensed at the nuclear membrane. Other H and SR cells were more similar to histiocytic cells or reticulum cells because of the large number of cell organelles (e.g., lysosome-like granules) and diffuse heterochromatin. Finally, cases of the nodular sclerosis type of Hodgkin's disease showed another cell type with some resemblance to IDC. The cells of this type are called lacunar cells because of their special light-microscopic appearance.Abbreviations H Hodgkin - IDC interdigitating reticulum cell - SR Sternberg-Reed     

Phenotypic analysis of nylon-wool-adherent suppressor cells that inhibit the effector process of tumor cell lysis by lymphokine-activated killer cells in patients with advanced gastric carcinoma

The causes of down-regulation of cytotoxic immune responses in cancer patients have not been fully evaluated. We previously demonstrated that T-cell-growth-factor-activated peripheral blood lymphocytes (PBL) with the surface phenotype CD8⁺ CD11b^–, from patients with widespread metastasis of gastric carcinoma, inhibited the effector process of lymphokine-activated-killer(LAK)-cell-mediated cytolysis. In this study, we examined suppressor cell activity in freshly prepared PBL from 18 patients with advanced gastric carcinoma, and 10 normal healthy individuals. The suppressor cell activity was assayed by recording whether or not PBL inhibited directly the effector process of LAK cell cytotoxicity. Most of the PBL suspensions from cancer patients showed that they contained a population of cells that can directly inhibit the effector phase of tumor cell lysis of the cytotoxic cells. To analyze further the PBL responsible for the suppression, the cells were passed over a nylon-wool column. Nylon-wool-adherent cells significantly augmented the suppression, while the cells passing through abrogated the suppressive effect. Most nylon-wool-adherent cells from 10 normal healthy controls did not inhibit the cytotoxic reaction. To determine further the suppressor-effector population in nylon-wool-adherent cells, negative-selection studies using CD8-, CD4- or CD11b-coated magnetic beads, and positive-selection studies using CD8- or CD4-coated magnetic beads were performed. Finally the results suggest that the suppressor-effector cells comprise at least two different surface phenotypes: CD8⁺ T and CD8^–CD11b⁺ cells. The possible role of CD4⁺ T cells and HLA-DR⁺ LeuM3⁺ macrophages as suppressor cells was ruled out in nylon-wood-adherent cells. CD8⁺ T and possibly CD8^–CD11b⁺ cells apparently suppressed the efferent limb of the antitumor immunity. The selective immune suppression mediated by these cells may partly be concerned with escape mechanisms of gastric carcinoma from the host immune surveillance system.Abbreviations TCGF T cell growth factor - PBL peripheral blood leucocytes - LAK lymphokine-activated killer - NK natural killer - CTL cytotoxic T lymphocytes     

Mesenchymal stem cells efficiently inhibit the proinflammatory properties of 6-sulfo LacNAc dendritic cells

Mesenchymal stem cells emerged as a promising treatment modality for steroid-refractory graft-versus-host disease, which represents a major complication of allogeneic hematopoietic stem cell transplantation. Dendritic cells (DCs) display an extraordinary capacity to induce T-cell responses and play a crucial role in the pathogenesis of graft-versus-host disease. Here, we investigated the impact of mesenchymal stem cells on the proinflammatory capacity of 6-sulfo LacNAc (slan) dendritic cells, representing a major subpopulation of human blood dendritic cells. Mesenchymal stem cells markedly impair maturation of slanDCs and their ability to secrete proinflammatory cytokines, which was dependent on prostaglandin E₂. In contrast, the release of anti-inflammatory IL-10 was improved by mesenchymal stem cells. Furthermore, mesenchymal stem cells efficiently inhibit slanDC-induced proliferation of CD4⁺ and CD8⁺ T cells and polarization of naïve CD4⁺ T lymphocytes into Th1 cells. These results indicate that mesenchymal stem cells significantly impair the high proinflammatory capacity of slanDCs and further substantiate their potential for the treatment of graft-versus-host disease.     

人尿源干细胞的分离培养及体外成神经细胞分化研究

目的从正常成人尿液中分离提取间充质干细胞(MSC)来源的人尿源干细胞(hUSC),并研究比较其在不同诱导液中的体外成神经分化能力。方法提取正常成人来源hUSC,经体外培养、扩增,流式细胞术鉴定,用4种不同配方的成神经诱导液分别诱导hUSC向神经细胞分化,在诱导至第14天时,光镜下观察细胞形态改变,并用逆转录-聚合酶链反应(RT-PCR)方法鉴定Nestin、S100、β3-tublin和NF-200基因表达。采用SPSS 13.0统计软件对数据进行分析。组间比较采用t检验和方差分析。结果从正常成人提取的hUSC成功表达MSC表面标志物(CD27~+、CD44~+、CD73~+、CD90~+、CD31~-、CD34~-、CD45~-、HLA-DR~-)。hUSC在B、C、D成神经诱导液中诱导培养14 d后,细胞胞体回缩饱满、折光度变强,呈现神经元样突起。相比A成神经诱导液,B、C和D成神经诱导液中细胞Nestin、S100表达水平增高,B和D成神经诱导液中细胞β3-tublin、NF-200表达水平降低。其中C成神经诱导液中细胞Nestin表达水平高于B和D成神经诱导液中的细胞,B成神经诱导液中细胞S100表达水平高于C和D成神经诱导液中细胞,差异均具有统计学意义(P0.05)。结论 hUSC具备MSC特性;B、C、D成神经诱导液均有助于hUSC向神经类细胞方向分化,提示在优化诱导条件下,hUSC能向神经干细胞(NSC)及胶质细胞亚群分化。     

树突状细胞与细胞融合后诱导T细胞介导的抗肿瘤效应

目的　研究树突状细胞与小鼠肥大细胞瘤细胞P815融合作为肿瘤疫苗免疫小鼠后抑制肿瘤生长的作用及其机理。方法　Balb/C小鼠DC与P815细胞体外融合 ,形成的杂交瘤分 2次免疫接种同基因小鼠皮下 ,然后用P815细胞攻击免疫的小鼠 ,观察肿瘤的生长情况及免疫小鼠脾细胞特异性CTL功能。结果　DC P815融合率为 3 0 %,杂交瘤接种小鼠能产生明显的抗肿瘤效应 ,其拮抗P815细胞攻击的能力明显强于用灭活的死P815细胞与DC混合 ,和单用死亡P815细胞免疫的小鼠及用生理盐水的对照组小鼠。DC P815杂交瘤免疫接种小鼠的脾细胞特异性CTL ,杀伤活性强于其它各组小鼠。结论　DC与肿瘤细胞融合后作为肿瘤疫苗接种可在体内产生明显的抗肿瘤免疫 ,其机理主要是特异性CTL的作用。     

动脉内注入内皮祖细胞促进血管内皮快速修复的实验研究

目的探讨体外培养的自体内皮祖细胞（EPC）对兔颈动脉球囊损伤后内皮修复以及内膜增生的影响。方法分离兔骨髓单个核细胞,EBM-2＋SingleQuots培养基培养,鉴定EPC表面标志,并标记。建立兔颈动脉球囊损伤模型,在动脉球囊损伤的即刻,将体外培养的自体EPC经损伤动脉缓慢注入动物体内。分别在手术后7、14天处死动物。检测损伤部位的内皮修复以及内膜增生情况,计算内膜中膜比值（I／M比值）并检测注入的细胞在血管损伤局部的分布。结果EPC治疗组在7、14天的内皮覆盖率分别为（50．923±2．476）％和（82．609±2．611）％,均明显高于对照组,差异有统计学意义。EPC治疗组14天的I／M比值为0．378±0．029,低于对照组,差异有统计学意义。免疫组织化学染色示EPC治疗组损伤血管局部有标记的EPC分布。结论内皮球囊损伤后动脉内注入EPC能有效促进内皮修复,减少内膜增生,预防再狭窄的发生。     

肺结核患者外周血记忆性T细胞及B细胞亚群的检测分析

目的探讨肺结核患者外周血记忆性T及B细胞亚群的表达水平。方法采用流式细胞仪检测肺结核患者（TB）、潜伏感染者（LTBI）、健康人（HD）外周血记忆性T、B细胞亚群水平及B细胞表达水平;利用Elispot技术检测外周血单个核细胞（PBMC）结核菌抗原特异性IFN-r分泌水平。结果流式结果显示,TB及LTB1的CD4＊及CD8＊中心记忆性T细胞（Tcm）显著增多,以CD4＊尤为显著;相反,效应记忆性T细胞（Tem）减少。TB记忆性B细胞均低于HD和LTBI（P〈0．01）;而在HD与LTBI及TB这三组CD19＆细胞占淋巴细胞比例中都有明显差异（P〈0．01;P〈0．001）。CD4’／CD8’Tcm与IFN-γ ELISPOT结果成正相关。结论肺结核患者外周血中存在着记忆性T及B细胞亚群的紊乱,中心记忆性T细胞增多,效应记忆性T细胞减少,记忆性T、B细胞的数量及免疫状态与结核菌感染的不同转归有关,记忆性T、B细胞在结核病发病机制中发挥着重要作用。