Expression and Content of Protein LC3B in Gastric Cancer Tissue,Relationship with Expression of mTOR,AMPK in Gastric Cancer Tissue and HER2 and PD-L1 Status of the Tumor

Bulletin of Experimental Biology and Medicine - We studied the mechanisms by which microRNA-126 regulates proliferation and migration of human umbilical vein endothelial cells (HUVEC) cultured in a...     

HESR1基因在血管新生中作用的初步研究

目的研究转录因子HESR1在血管新生中的作用。方法检测内皮细胞激活状态HESR1表达的影响,克隆HESR1基因,转染到HUVEC,绿色荧光和PCR观察HESR1在内皮细胞的表达,流式细胞仪检测它对血管内皮细胞增殖,boyden小室检测对细胞迁移的影响,建体外二维血管模型,观察HESR1对血管形成的影响。结果内皮细胞激活状态HESR1的表达下降,HESR1基因能抑制内皮细胞的增殖和迁移,减少血管新生。结论HESR1基因通过抑制内皮细胞的增殖和迁移,使内皮细胞从激活状态转入安静状态,减少血管的形成,维持血管的稳定状态。     

siRNA沉默FOXO3a对三氧化二砷抑制内皮细胞增殖的影响

背景：课题组前期已证实As2O3可以促进乳腺癌细胞中FOXO3a因子的表达,并抑制肿瘤细胞中血管内皮生长因子的表达,但As2O3对血管内皮细胞增殖的影响,以及对血管内皮细胞中FOXO3a、血管内皮生长因子的影响尚未证实。 目的：观察siRNA沉默FOXO3a对As2O3抑制人脐静脉内皮细胞增殖及血管生成的影响。 方法：实验分为4组：①As2O3组：待人脐静脉内皮细胞贴壁,在含4 μmol/L As2O3的RPMI-1640培养基中培养。②control siRNA组：转染无关序列control siRNA后,细胞在含4 μmol/L As2O3的RPMI-1640培养基中培养。③FOXO3a siRNA组：转染FOXO3a siRNA后,细胞在含4 μmol/L As2O3的 RPMI-1640培养基中培养。④对照组：与实验药物等体积PBS作为对照,细胞在完全培养基条件下培养。应用CCK-8方法检测细胞增殖情况,采用免疫细胞化学方法观察人脐静脉内皮细胞中FOXO3a、血管内皮生长因子蛋白的表达情况。 结果与结论：与对照组相比,FOXO3a siRNA明显减弱了As2O3抑制人脐静脉内皮细胞增殖的作用,As2O3促进人脐静脉内皮细胞中FOXO3a蛋白的表达并抑制血管内皮生长因子蛋白的表达,FOXO3a siRNA处理后明显抑制FOXO3a蛋白表达且增加血管内皮生长因子蛋白表达。结果提示FOXO3a可能成为As2O3抑制肿瘤细胞增殖及血管生成治疗的重要靶点。     

Vascular endothelial cells provide T cells with costimulatory signals via the OX40/gp34 system

We investigated whether gp34, the ligand of OX40, expressed on EC is involved in costimulation of T cells. Normal CD4+ T cells were stimulated with anti-CD3-coated beads, phytohemagglutinin (PHA), or concanavalin A (Con A) in the presence or absence of irradiated human umbilical vein endothelial cells (HUVEC). Stimulation of T cells with each of these mitogens results in significant T-cell proliferation only when HUVEC were present, and this proliferation was inhibited markedly by anti-OX40 or anti-gp34 monoclonal antibody (mAb). T cells cultured with HUVEC produced more interleukin (IL)-2 than those cultured without HUVEC. The addition of anti-IL-2R alpha chain and anti-IL-2R beta chain mAbs abolished the costimulatory effects of HUVEC. Thus, the augmentation of T-cell proliferation appears to be attributable to increased IL-2 production. These results suggest that gp34 expressed on HUVEC plays a role in potentiation of T-cell immune response by providing OX40+ T cells with costimulatory signals.     

The impact of proliferative potential of umbilical cord-derived endothelial progenitor cells and hypoxia on vascular tubule formation in vitro

Revascularization of the damaged tissue is pivotal to tissue repair. Here, by bringing together two in vitro model systems, we have been able to examine (1) the ability of human umbilical vein endothelial cells (HUVEC) containing a complete hierarchy of endothelial progenitors derived from the human umbilical cord to generate vascular tubules within a human stromal niche in vitro and (2) the effects of exposure to low oxygen tensions on endothelial progenitor cell proliferation and tubule formation in vitro. Our results demonstrate that high proliferative potential endothelial colony forming cells (HPP-ECFC) from cultured HUVEC preferentially contribute to vascular tubule formation in vitro and that these progenitor cells are concentrated in the CD34(lo/-) fraction. HUVEC were initially resistant when exposed to hypoxia (1.5% O(2)) for short periods (1-2 days), but sustained chronic hypoxia (4-14 days) inhibited their ability to proliferate. This was reflected by a loss in their ability to form tubules in cocultures of human dermal fibroblasts (hDFs). In contrast, an acute exposure to low oxygen tensions (1.5% O(2) for 24 h) followed by reoxygenation did not adversely affect the capacity of these cells to both proliferate and form vascular tubules in vitro.These studies therefore provide a model system to study the influences of the microenvironmental niche and modification of this niche on vascular tubule formation in vitro from HPP-ECFC.     

葛根素通过ERK1/2信号通路促进内皮细胞增殖

目的:体外观察葛根素对人脐静脉内皮细胞(HUVEC)的促增殖作用,并对其机制进行初步探讨。方法:采用四甲基偶氮唑蓝(MTT)法检测不同浓度葛根素对HUVEC细胞增殖的影响;流式细胞术检测细胞凋亡;Western blot检测p-ERK1/2蛋白表达水平。结果:葛根素能剂量依赖性促进HUVEC增殖,在80μM效果最明显,且作用36h时,能显著减少早期凋亡,激活ERK1/2磷酸化。结论:葛根素素对人脐静脉内皮细胞有明显的促进增殖作用,其作用可能与ERK1/2的活化有关。     

Induction of contact-dependent endothelial apoptosis by osteosarcoma cells suggests a role for endothelial cell apoptosis in blood-borne metastasis

Although tumour cells are believed to migrate between endothelial cells early in metastasis, the possibility remains that endothelial apoptosis may also contribute to the tumour's breach of the vascular barrier. Although seemingly inconsistent with tumour angiogenesis, one publication describes the induction of contact-dependent apoptosis in cultured endothelium by tumour cells. The cell culture data are, however, open to challenge on technical grounds while there are no confirmatory reports. The present paper describes experiments overcoming these limitations. SAOS-2 human osteosarcoma cells and two rat carcinoma cell lines were co-cultured with human umbilical vein endothelial cells (HUVECs) and cultures labelled by surface lectin histochemistry for endothelium. The HUVEC culture density was determined and SAOS-2 cells, but not rat carcinoma cells, were found significantly to reduce HUVEC survival despite the release of potent growth factors as determined in separate experiments with tumour cell conditioned medium. Lectin labelling combined with light microscopy, transmission electron microscopy, flow cytometry for both lectin binding and DNA content, and DNA gel electrophoresis of SAOS-2/HUVEC co-cultures revealed extensive HUVEC apoptosis. These findings indicate contact-dependent endothelial apoptosis by SAOS-2, while this activity appeared weaker and overwhelmed by HUVEC proliferation with rat carcinoma cells. Importantly, this study supports the suggestion that endothelial apoptosis may be important for metastasis and suggests a complex interplay between endothelial proliferation and apoptosis in tumours.     

A Modular Tissue Engineering Construct Containing Smooth Muscle Cells and Endothelial Cells

Human umbilical vein endothelial cells (HUVEC) were seeded on sub-mm sized collagen cylinders containing embedded umbilical vein smooth muscle cells (UVSMC). These cylindrical “modules” are intended to be used as a vascularized construct, in which HUVEC lined channels are created by the random packing of the modules in situ or within a larger container. Embedding UVSMC cultured in medium containing 10% FBS had an adverse effect on subsequently seeded HUVEC junction morphology; HUVEC/UVSMC co-culturing was done in HUVEC medium (2% FBS with the addition of 0.03 mg/mL endothelial cell growth supplement) as compared to HUVEC seeded on collagen-only modules. In contrast, embedding UVSMC cultured in serum-free medium prior to embedding improved EC junction morphology. Such serum-free culturing, also prevented the UVSMC induced contraction of the collagen modules. On the other hand, embedding serum-free cultured UVSMC promoted HUVEC proliferation and NO secretion compared to those modules embedded with 10% serum cultured UVSMC. These results suggest, not surprisingly, that embedded UVSMC phenotype plays an important role in the seeded HUVEC phenotype, and that the response can be modulated by the UVSMC culture medium serum concentration. These studies were undertaken with a view to using the UVSMC to modulate the thrombogenicity of the HUVEC. Exploration of this outcome awaits further studies directed to understanding the mechanism of the cellular interactions.     

鼠内皮抑素基因腺病毒载体的构建及对血管内皮细胞的生长抑制

构建含鼠内皮抑素基因的腺病毒载体（pAd／mEnd），并观察内皮抑素蛋白对人脐静脉血管内皮细胞（HUVEC）的生长抑制、细胞周期和凋亡的影响。结果发现pAd／mEnd感染BEL7402细胞后，能有效表达内皮抑素蛋白，显著抑制HUVEC增殖，阻滞HUVEC细胞S期，凋亡指数显著增加。     

An improved endothelial barrier model to investigate dengue haemorrhagic fever

A cell culture model suitable for studies of dengue haemorrhagic fever was developed, based on culture of primary human umbilical vein endothelial cells (HUVECs) on a permeable membrane. By electron microscopy, cultured HUVECs at day 11 resembled morphologically microvascular endothelium. Endothelial barrier function was assessed by measuring transendothelial flux of albumin. Instead of using a labelled tracer molecule, an enzyme-linked immunosorbent assay (ELISA) was developed to measure concentrations of native human albumin. The permeability characteristics of the HUVEC monolayer were found to be improved significantly (approximately 1 log reduction in permeability coefficient for albumin) by culturing HUVECs in human serum rather than fetal calf serum. Permeability coefficients for albumin in the range 1-4 x 10(-7) cm/s were achieved, which is an improvement on previous in vitro models of the endothelial barrier. Comparison of transendothelial flux of albumin and urea provided evidence of molecular sieving by the HUVEC monolayer. Moreover, tumour necrosis factor-alpha induced a dose-dependent, reversible increase in permeability of the HUVEC monolayer. This endothelial barrier model thus has many important characteristics that resembled human microvascular endothelium and is an improvement on the previous model proposed for studies of dengue haemorrhagic fever.     

Functional polymorphism of IgG FcRII (CD32) on human neutrophils.

In this study we demonstrate that Fc gamma RII on polymorphonuclear cells (PMN) (i) mediates binding of immune complexes, and (ii) is polymorphic, similar to the polymorphism described for monocytes and platelets. This was demonstrated in an adherence assay of PMN to activated human umbilical vein endothelial cells (HUVEC). Precoating of activated HUVEC with a mouse IgG1 monoclonal antibody (mAb) ENA1, which is highly reactive with activated HUVEC, caused an enhanced adhesion in 11 of 15 experiments using PMN from different donors. Enhanced adhesion of the PMN corresponded with expression of a high-responder (HR) Fc gamma RII on monocytes isolated from the same donor, as identified by anti-CD3-induced T-cell proliferation. These data led to the conclusion that Fc gamma RII on PMN is polymorphic. This conclusion was, furthermore, supported by immunofluorescence (IF) studies using a new mAb 41H16, which selectively reacts with the HR allotype of Fc gamma RII.     

多聚赖氨酸修饰钛氧膜并固定纤连蛋白对内皮细胞生长的影响

为了进一步提高钛氧膜与内皮细胞的亲和性,研究了一种表面生物化学方法对钛氧膜表面内皮细胞生长的影响.先利用盐酸和双氧水活化钛氧膜表面,使其产生羟基基团,再涂覆多聚赖氨酸作为中间过渡层,最后在表面上固定纤连蛋白.采用傅立叶红外光谱仪、X射线光电子能谱仪及接触角测量的方法对每步处理后的材料表面进行分析与测试.通过体外人脐静脉原代内皮细胞培养实验评价内皮细胞在样品表面的黏附和生长.研究结果显示,通过表面活化后涂覆多聚赖氨酸并固定纤连蛋白的方法能促进内皮细胞在钛氧膜表面的黏附和生长.     

Interleukin-1 production and cell-activation response to cytomegalovirus infection of vascular endothelial cells

Summary Human cytomegalovirus (HCMV) is a source of major complications in immunosuppressed individuals, and endothelial involvement in HCMV infection is well documented. Traditionally laboratory strains of HCMV have been used in experimental investigations in vitro; however the continuous propagation of these strains in fibroblasts have attenuated the virus making it unsuitable for infecting other cell systems such as endothelial cells. In this study a recent clinical isolate of HCMV was propagated through several passages in endothelial cells and was used to investigate the effect of HCMV infection of human umbilical vein endothelial cells (HUVEC) on IL-1 production and cell proliferation. Infection of HUVEC with the clinical isolate of HCMV (at multiplicity of infection 5:1) suppressed the production of IL-1 alpha (82%) and IL-1 beta (99%) at 5h post infection; the levels returned to that of the control within 24h post infection. Ultraviolet inactivated (but not heat killed) virus produced similar suppression confirming that a thermolabile viral structural protein or intact virion were responsible for this suppression. Infection of HUVEC with the clinical isolate increased the number of these cells and the rate of their proliferation. An increase of infected HUVEC number under quiescent growth conditions continued as the infection progressed (6–10 days post infection), exhibiting, at 3 days post infection, 5 times the number of uninfected HUVEC (control) which did not tolerate the quiescent culture conditions for more than 4 days. Live virus is responsible for this increase because UV-inactivated virus did not maintain the proliferation of HUVEC. These studies suggest that while infection of HUVEC with a recent clinical isolate of HCMV suppressed the production of IL-1 at early hours after infection, it increased the proliferation of these cells at later stages of infection.     

泰素抑制内皮细胞管道组装功能的机理研究

目的体外实验研究显示低浓度泰素具有抑制血管内皮细胞组装形成管道的能力。本研究探索泰素这种作用的机理。方法体外培养的人脐静脉血管内皮细胞(HumanUmbilicalVeinEn-dothelialCell,HUVEC)加入低浓度泰素,观察HUVEC的细胞形态,荧光染色检测HUVEC细胞内actin的表达情况。在体外管道形成试验中检测抗TLR-4(Tolllikereceptor-4)抗体对泰素抑制HUVEC管道组装能力的拮抗作用。结果实验结果发现低浓度泰素可使体外培养的HUVEC形态向间质细胞转化。荧光染色发现泰素可抑制HUVEC细胞actin表达。而对其他细胞无类似作用。采用TLR-4抗体可拮抗泰素抑制HUVEC管道形成的作用。结论低浓度泰素通过TLR-4抑制HUVEC细胞内actin的表达,使内皮细胞形态向间质细胞转化,从而抑制内皮细胞组装血管的能力。泰素的这种作用具有血管内皮细胞特异性。     

Effect of a specific cysteinyl leukotriene-receptor 1-antagonist (montelukast) on the transmigration of eosinophils across human umbilical vein endothelial cells

BACKGROUND: Leukotrienes have been implicated in the selective infiltration of eosinophils into the bronchial mucosa in asthma. OBJECTIVE: We studied whether eosinophil transmigration through cultured human umbilical vein endothelial cells (HUVECs) can be blocked by a specific cysteinyl LT1-receptor-antagonist. METHODS: Unstimulated and stimulated eosinophils from patients with asthma and normal controls were subjected to confluent human umbilical vein endothelial cell (HUVEC) monolayers separating the upper and lower chamber of Transwell culture plates. Unstimulated eosinophils or cells pre-incubated in the presence of the eosinophil activating cytokines GM-CSF or IL-13 were placed in the upper chambers while PAF, a potent chemoattractant factor for eosinophils, was added to the lower chamber. Migration of eosinophils was quantified by a beta-glucuronidase assay. RESULTS: The assumption that eosinophils express CysLT1 (cysteinyl-leukotriene 1)-receptors was based on our demonstration of mRNA-expression for the CysLT-1-receptor by polymerase chain reaction on purified eosinophils. The chemotactic response to PAF was significantly reduced when eosinophils were pre-incubated with montelukast for 15 min. When eosinophils were pre-incubated with GM-CSF and/or IL-13, the migratory response to PAF was also significantly reduced by montelukast. CONCLUSION: From these data we conclude that the specific cysteinyl LT1-receptor antagonist montelukast can inhibit PAF-induced eosinophil transmigration through cultured HUVEC monolayers.     

Anti-CD14 antibodies reduce responses of cultured human endothelial cells to endotoxin.

Lipopolysaccharide (LPS) activates both myeloid and endothelial cells. Whereas CD14 has been shown to be involved in LPS recognition by myeloid cells, the mechanism responsible for the strong response of endothelial cells to LPS remains to be elucidated. The role of CD14 in this process was studied using CD14-specific antibodies (Ab). Anti-CD14 Ab inhibited LPS-induced interleukin-6 (IL-6) release and E-selectin expression by cultured human umbilical vein endothelial cells (HUVEC). Messenger RNA encoding IL-6 and E-selectin was reduced in parallel. The inhibitory effect of anti-CD14 Ab was epitope dependent, maximal at low LPS concentrations and dropping with increasing LPS doses. Anti-CD14 Ab did not affect endothelial cell activation induced by IL-1 beta, tumour necrosis factor-alpha (TNF-alpha) and phorbol 12-myristate 13-acetate (PMA). IL-6 release and E-selectin expression of HUVEC were strongly reduced when LPS activation was performed in the absence of serum, indicating involvement of serum components in LPS activation of HUVEC. Nevertheless, anti-CD14 Ab also blocked LPS-induced HUVEC activation in the absence of serum. Although the role of serum components in LPS activation remains to be elucidated, CD14 seems to be a key mediator in LPS-induced activation of endothelial cells.     

胰蛋白酶消化法体外培养和鉴定人脐静脉血管内皮细胞*☆

背景：体外建立稳定可靠的人脐静脉血管内皮细胞模型,目前培养方法缺乏统一的标准。 目的：探索脐静脉内皮细胞的体外培养的方法。 方法：采用2.5 g/L胰蛋白酶和0.02%EDTA消化、分离、体外原代培养及消化传代脐静脉内皮细胞,简化完全培养液组分(不添加血管内皮细胞生长因子、肝素等辅助因子),当原代培养细胞80%以上汇合,根据细胞特有的形态学特征和Ⅷ因子进行内皮细胞的鉴定。 结果与结论：种植在培养瓶中的内皮细胞2 h贴壁生长,24 h换液后内皮细胞80%融合,细胞状态好,内皮细胞呈单层铺路石样外观,经过镜下观察和Ⅷ因子相关抗原鉴定证明是脐静脉内皮细胞。证实用胰蛋白酶灌注脐静脉是一种简单、实用的获得人脐静脉血管内皮细胞的方法,可靠性大,成功率高,可以构建体外研究血管内皮细胞的模型。关键词：人脐静脉;血管内皮细胞;细胞培养;形态学;鉴定 缩略语注释：HUVECs: human umbilical vein endothelial cells,人脐静脉血管内皮细胞 doi:10.3969/j.issn.1673-8225.2012.15.008     

The effect of human osteoblasts on proliferation and neo-vessel formation of human umbilical vein endothelial cells in a long-term 3D co-culture on polyurethane scaffolds

Angiogenesis is a key element in early wound healing and is considered important for tissue regeneration and for directing inflammatory cells to the wound site. The improvement of vascularization by implementation of endothelial cells or angiogenic growth factors may represent a key solution for engineering bone constructs of large size. In this study, we describe a long-term culture environment that supports the survival, proliferation, and in vitro vasculogenesis of human umbilical vein endothelial cells (HUVEC). This condition can be achieved in a co-culture model of HUVEC and primary human osteoblasts (hOB) employing polyurethane scaffolds and platelet-rich plasma in a static microenvironment. We clearly show that hOB support cell proliferation and spontaneous formation of multiple tube-like structures by HUVEC that were positive for the endothelial cell markers CD31 and vWF. In contrast, in a monoculture, most HUVEC neither proliferated nor formed any apparent vessel-like structures. Immunohistochemistry and quantitative PCR analyses of gene expression revealed that cell differentiation of hOB and HUVEC was stable in long-term co-culture. The three-dimensional, FCS-free co-culture system could provide a new basis for the development of complex tissue engineered constructs with a high regeneration and vascularization capacity.     

Endothelial cell growth factor (ECGF) enmeshed with fibrin matrix enhances proliferation of EC in vitro.

The vascular biomaterials that are currently used for clinical implants have been considered as poor substrates for human endothelial cell adhesion and spreading. Therefore, thrombotic occlusion is the predominant cause for the failure of small diameter vascular grafts made out of Dacron or Teflon. To reduce surface thrombogenicity of material surfaces used for vascular implants, in vitro seeding of endothelial cells using adhesive protein matrix is under evaluation in various laboratories. Evidences suggest that fibrin matrix is a suitable matrix for endothelial cell (EC) adhesion to the currently available vascular graft materials; however, poor proliferation of attached cells seems to be a major limitation. During this study we have also found that fibrin is a better matrix compared to gelatin to support cell attachment and spreading. However, the poor proliferation of initially attached human umbilical cord vein endothelial cell (HUVEC) necessitated modification of the matrix composition to get a monolayer within a limited period. Since fibrin can form a network of protein bundles, an effort is made to incorporate growth factors within the matrix. Endothelial cell growth factor (ECGF) isolated from bovine hypothalamus is immobilized on the surface with fibrin glue (FG) to promote proliferation of HUVEC. The results demonstrate that proteins with similar molecular weights as growth factors (GF) are retained within the matrix and released into the culture medium for 96 h, in quantities that would be sufficient to promote cell proliferation. When cells were seeded on the matrix composed with components of FG and ECGF, the HUVEC proliferated at a significantly higher rate compared to the cells on surfaces coated with gelatin or fibrin. The EC thus grown on the composite (FG + ECGF) resisted the shear stress as compared to the cells grown on gelatin. The HUVEC monolayer grown on the composite seems thromboresistant as adhesion and activation of platelets are negligible after platelet rich plasma is incubated with the monolayer for about 1 h with agitation. Therefore, the composite of fibrin and ECGF can be a suitable matrix for further evaluation of patients' autologous endothelial cell attachment and proliferation for clinical application.     

重组人钙调素对人脐静脉内皮细胞增殖作用的影响

本文探讨外源性重组人钙调素(recombinant human calmodulin,rhCaM)对人脐静脉内皮细胞(HUVEC)增殖作用的影响。采用基因重组技术在大肠杆菌DH5α中高效表达并纯化rhCaM。将HUVEC接种于96孔培养板,加入不同浓度rhCaM,用MTT比色法检测rhCaM对HUEVC细胞增殖作用的影响。结果表明rhCaM在0.04～0.4μg/ml浓度范围内可促进HUEVC的增殖,促增殖作用随细胞培养密度的增加而减弱,也与培养基中新生小牛血清的含量密切相关。故rhCaM对HUEVC增殖具有促进作用。