脂肪组织来源干细胞治疗缺血性心脏病的研究进展

干细胞移植治疗心血管疾病尤其是缺血性心脏病取得了令人鼓舞的结果,但既往研究主要集中于胚胎干细胞和骨髓间充质干细胞的研究。近年来,人们发现脂肪组织来源干细胞（ASCs）与其他组织来源干细胞相比具有来源广、取材创伤小、具有多向分化潜能、增殖能力强等优点,故近几年来ASCs在心血管疾病治疗上的作用受到越来越多的重视。本文对ASCs细胞生物学特性以及ASCs作为种子干细胞在缺血性心脏病、心肌细胞再生方面的基础和临床研究进展作一综述。     

未来肝癌治疗的新靶点-肝癌干细胞

肿瘤起源于干细胞的假说正在各种人类肿瘤中得到证实,肿瘤不单是一种基因病,而且是一种干细胞病.基因突变作用于干细胞,干细胞突变成为肿瘤干细胞,这是肿瘤发生、再生、转移和复发的关键.最新研究表明,肝细胞型肝癌可能是由肝干细胞未分化或分化不全引起的.对于肝癌细胞的研究我们知道肝细胞型肝癌中的细胞具有干细胞特性,如永生性,可移植性以及对治疗手段的抵抗性.但至今为止,确切的肝癌干细胞的标志物没有找到,而且没有分离出肝癌干细胞.     

肝癌干细胞的研究现状

传统的理论认为肿瘤的生长是所有肿瘤细胞共同增殖的结果。癌干细胞假说的提出使肿瘤越来越被设想为一种干细胞疾病^[1]。这种假说认为肿瘤的生长是由一小部分肿瘤干细胞增殖的结果，这些细胞具有干细胞特性，尤其是具有自我更新、产生和维持肿瘤生长和导致细胞异质性的原始细胞及分化细胞的能力。肝癌是最常见的一种肿瘤，是否存在肝癌干细胞一直是备受关注。本文介绍有关肝癌干细胞的研究现状。     

间充质干细胞向肝细胞诱导分化的研究进展

间充质干细胞（mesenchymal stem cells,MSCs）作为一种多能干细胞,具有横向分化能力,在组织工程、基因工程以及临床应用上有着巨大的应用价值。我国是肝病大国,肝硬化、肝癌和肝衰竭已成为众多肝病患者的最终归宿。干细胞移植为治疗终末期肝病提供了新的希望,而间充质干细胞以其独特的优势,得到许多研究者的青睐。     

心肌梗死后骨髓间充质干细胞移植对心室肌细胞复极活动的影响

目的调查心肌梗死后植入骨髓间充质干细胞（mesenchymal stem cells，MSCs）对心室肌细胞复极活动的影响。方法10只健康幼猪作为正常对照组，23只幼猪通过球囊导管堵闭左冠状动脉前降支法建立心肌梗死模型并分别移植MSCs悬液（干细胞组，n=13）或等量生理盐水（梗死组，n=10），6周后行心肌组织免疫组化双染色、左心室血流动力学及左心室肌细胞复极活动的检查。结果（1）心肌组织免疫组化双染色示，MSCs源性细胞集中分布于梗死区，胞浆中出现了肌钙蛋白T（troponinT）；（2）干细胞组左心室射血分数、左心室收缩压和舒张末压虽未恢复正常（与对照组相比，P均〈0．01），但较梗死组均改善（P均〈0．05）；（3）梗死组和干细胞组动作电位复极90％的时间（APD90）、复极时间（RT）、APD和RT离散度值较对照组均延长（P均〈0．01），但干细胞组较梗死组显著缩短（P〈0．05—0．01）；（4）对照组APD重建曲线斜率〈1（正常），而干细胞组和梗死组APD重建曲线斜率均〉1（异常），但前者斜率明显小于后者；（5）干细胞组诱发APD交替的阈值周长虽高于对照组（P〈0．01），却低于梗死组（P〈0．05）。结论MSCs移植可减轻梗死心室复极紊乱，其机制可能与MSCs参与心肌组织修复、改善血流动力学水平有关。     

人间充质干细胞治疗肝病研究进展及前景

间充质干细胞（MSCs）是干细胞的一类，因能分化为间质组织得名，是属于中胚层的一类多能干细胞，主要存在于结缔组织和器官间质中，骨髓中含量最丰富。MSCs可以向多种中胚层和神经外胚层来源的组织细胞分化，可分化为成骨细胞、软骨细胞、脂肪细胞、肌腱细胞、平滑肌细胞、骨髓基质细胞、成纤维细胞及多种血管内皮细胞，甚至神经系统的神经元和神经胶质细胞，使之成为治疗多系统疾病的“实用型于细胞”。     

骨髓间充质干细胞及基因治疗心血管疾病的研究进展

骨髓间充质干细胞（mesenchymal stem cells，MSCs）是存在于骨髓中，除造血干细胞外的另一类早期前体细胞；其来自基质，也称骨髓基质干细胞。1987年Friedenstein等在分离大鼠骨髓成骨祖细胞时，首次从骨髓基质中鉴定出一种非造血系成体多能干细胞，并实验证实在体外这类细胞能分化成各种间质细胞，包括成骨细胞、脂肪细胞、软骨细胞和肌肉细胞等，因而称之为MSCs。     

大鼠骨髓间充质干细胞在急性肝损伤环境中的分化

骨髓中除造血干细胞（HSCs）外，还存在着另外一类干细胞，即间充质干细胞（MSCs）。目前研究表明，MSCs具有横向分化能力，尤其能诱导分化为肝细胞，成了肝干细胞研究的热点，也为临床治疗终末期肝病提供了新思路。目前报道肝脏疾病影响17．5％的人群，终末期肝病的治疗在临床尚没有可行的办法。因此，迫切需要建立一种更有效的治疗方法。本研究探讨了骨髓MSCs在急性肝损伤环境中的分化。     

间充质干细胞在再生障碍性贫血发病机制中作用的研究

目的探讨再生障碍性贫血（AA）患者的骨髓间充质干细胞（MSCs）体外对T淋巴细胞的免疫调节作用。方法分离和体外培养骨髓间充质干细胞，与植物血凝素刺激的T细胞共培养，流式细胞仪检测T细胞的表面活化标志，MTT法检测T细胞受抑情况。结果AA患者骨髓MSCs在细胞形态和免疫表型上与缺铁性贫血患者没有明显差异，但对活化T细胞的免疫抑制作用明显减弱，而且SAA与CAA患者的骨髓MSCs之间也存在明显差异。结论AA患者骨髓MSCs对T细胞的免疫抑制作用减弱。     

左卡尼汀提高大鼠心肌梗死后移植骨髓间充质干细胞的存活和作用

目的探讨左卡尼汀（LC）是否能提高心肌梗死后移植骨髓间充质干细胞（MSCs）的存活率,从而提高其疗效。方法60只大鼠随机分为5组：假手术组、模型组、LC组、MSCs组和左卡尼汀联合骨髓间充质干细胞（LC＋MSCs）组。4周后检测心功能、MSCs存活情况和梗死区心肌纤维化水平。结果4周后,与MSCs组比较,LC＋MSCs组MSCs的存活、左室心功能、心肌纤维化均改善（P〈0．05）。结论LC联合MSCs使用可提高MSCs的存活率,更好地改善心肌梗死后的心功能和心肌纤维化。     

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.     

CD5 positive immunoregulatory B cell subsets

B-chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease often expressed as a clonal expansion of CD5+ B cells. We report the characterization of CD5+ B cells from two unique B-CLL patients. Cells from patient 1 coexpressed CD5 (leu-1), CD19 (Leu-12), CD20 (B1), and HLA-DR; they were CD10 (J5), CD21 (B2), CD22 (Leu-14), CD25 (IL2-R1), PCA-1, surface, and cytoplasmic Ig negative. They suppressed normal peripheral blood lymphocyte (PBL) pokeweed mitogen (PWM) -stimulated immunoglobulin (Ig) synthesis greater than 80%. Cells from patient 2 were CD5 (Leu-1), CD19 (Leu-12), CD20 (B1), CD21 (B2), CD22 (Leu-14), HLA-DR, IgM, and kappa positive. They were negative for CD10 (J5), CD25 (IL2-R1), and PCA-1. These cells did not suppress normal PBL PWM-stimulated Ig synthesis but produced a monoclonal IgM kappa protein with rheumatoid factor-like activity. These observations suggest that there are different CD5+ B cell subsets, one immunosuppressive and the other autoreactive.     

壁层上皮细胞的研究

近年来,随着对壁层上皮细胞(parietal epithelial cells,PECs)认识的加深,发现PECs发挥着重吸收蛋白、滤过屏障等重要功能,而且在肾脏受到损伤时,PECs可能作为潜在的干细胞,促进肾脏修复。本文就PECs及相关进展作一综述。     

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。     

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.     

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。     

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     

Transplanted human bone marrow contributes to vascular endothelium

Recent evidence indicates that bone marrow is a source of endothelial progenitor cells that are mobilized into the peripheral blood in response to cytokines or tissue injury. Previously, we showed that functional endothelial cells (ECs) can be clonally derived from phenotypically defined hematopoietic stem cells. To determine the EC potential of human bone marrow and peripheral blood stem cells, blood vessels in sex-mismatched transplant recipients were evaluated. EC outcomes were identified by using a combination of immunohistochemistry and XY interphase FISH. Donor-derived ECs were detected in the skin and gut of transplant recipients with a mean frequency of 2% and could readily be distinguished from CD45-expressing hematopoietic stem cells. None of the >4,000 ECs examined had more than two sex chromosomes, consistent with an absence of cell fusion. Y chromosome signals were not detected in sex-matched female recipients, excluding the vertical transmission of male cells. None of the recipients evaluated before hematopoietic engraftment demonstrated donor-derived ECs, indicating a close linkage between the recovery of hematopoiesis and EC outcomes. Transplantable bone marrow-derived endothelial progenitor cells may represent novel therapeutic targets for hematopoietic and vascular disease.     

脐血、外周血内皮祖细胞分化成内皮细胞的实验研究

目的探讨人的脐血、外周血内皮祖细胞（endothelialprogenitorcells,EPCs）体外分离、纯化、诱导扩增和分化为内皮细胞的可行性,并检测其表型和功能。方法新鲜脐血和健康成年人的外周血,使用Ficoll密度梯度离心法得单个核细胞,在M199培养基中体外培养,3d后去除悬浮细胞,继续培养,诱导EPCs增殖和分化。流式细胞仪检测EPCs标志CD34和内皮细胞特异性标志CD31表型,RTPCR检测ecNOS,flk1/KDR基因水平表达,免疫组化验证蛋白水平表达,并进一步通过NO活性的变化检测内皮细胞的功能。结果流式细胞仪检测,外周血单个核细胞（peripheralbloodmononuclearcells,PBMC）刚分离时,CD34阳性表达率为（1.1±0.8）%,培养3d后为（16.9±6.2）%。细胞形态观察发现,刚分离的单个核细胞呈圆形,形态小,3d后有明显集落形成,7d后梭形细胞线样排列,随培养时间增加,细胞形态逐渐变大,呈现出典型铺路石样改变。脐血单个核细胞（umbilicalcordbloodmononuclearcells,CBMC）和PBMC培养10d后,CD31阳性表达率分别为（76±17）%和（82±9）%。RTPCR检测有内皮细胞特异性成分ecNOS,flk1/KDR的表达。免疫组化染色,细胞膜和细胞浆中有弥漫性棕色出现,呈阳性反应,证实了蛋白水平的表达。培养10d的贴壁细胞随着VEGF浓度增加,NO生成增加,具有内皮细胞的功能。结论脐血,外周血EPCs体外分离,纯化,诱导培养后的贴壁细胞表型检测,大部分细胞具有内皮系标志物,并具有产生NO功能。