Human endothelial cell proliferation inhibiting activity in the sera of patients suffering from 'shock' or 'sepsis'

Abstract. The response of DNA-synthesis of human endothelial cells to sera derived from twenty-five patients suffering from 'sepsis' or 'shock' was measured by autoradiographic methods. In eight cases a constant decrease in proliferative response was found compared to that of sera from healthy donors. These proliferation values were shown to lie below the '60%-of-control-line'. The difference between the means of control and of corresponding 'low-response' values was significant ( P <0.05). In three cases a diminished response was caused only by some of several serum samples taken at different times. These results correlated well with the clinical state and outcome of patients but not with any of the over sixty clinical, therapeutic, laboratory and post-mortem parameters of investigation. Evidence is presented for a proliferation inhibiting activity in sera of patients in clinically poor states, and some physico-chemical properties of this 'factor' are described. Lethal injury to the cells or an impairment of cellular migration could not be observed within the observation periods used in this study.     

Human endothelial cells regulate survival and proliferation of human mast cells

Mast cells (MCs) are immunoregulatory and inflammatory tissue cells preferentially located around blood vessels. Since endothelial cells have been suggested to regulate MC functions, we analyzed MC-endothelial cell interactions in vitro by performing coculture experiments with purified human intestinal MCs and human umbilical vein endothelial cells (HUVECs). We found that HUVECs provide signals allowing MCs to survive for at least 3 wk and to proliferate without addition of cytokines; otherwise all MCs died. HUVEC-dependent MC proliferation was more pronounced than that induced by stem cell factor (SCF), known to act as an MC growth factor both in vitro and in vivo. After coculture with HUVECs, most MCs were of the tryptase and chymase double-positive phenotype (MC(TC)). Transwell experiments suggested that the HUVECs' effects on MCs are not mediated by soluble factors. HUVEC-dependent MC adhesion and proliferation were inhibited by neutralizing antibodies directed against SCF and vascular cell adhesion molecule (VCAM)-1 expressed on HUVECs, and c-kit and very late antigen 4 (VLA-4) on MCs. The data suggest that two mechanisms (membrane-bound SCF/c-kit and VCAM-1/VLA-4) are involved in human MC-endothelial cell interactions. In conclusion, our study provides evidence that endothelial cells regulate MC survival and preferentially support human MC(TC) development.     

Chorionic gonadotropin-induced cell proliferation and polyclonal immunoglobulin synthesis in human mononuclear cells

Some fetal and placental proteins may play a role in stimulating normal and malignant cell proliferation. We studied the effect of human chorionic gonadotropin (hCG) on the proliferative response of mitogen or alloantigen-activated, human peripheral blood mononuclear cells (PBM). The effect of hCG on polyclonal immunoglobulin synthesis was determined in pokeweed mitogen-activated cultures of PBM. hCG had a statistically discernible, augmenting effect on thymidine uptake in lectin-stimulated mononuclear cell cultures. This effect appeared to be dose-dependent, with an optimal range at 50-150 ng/hCG/ml. Polyclonal IgG and IgM synthesis was also significantly increased, both in PWM-stimulated and PWM-free cultures of PBM. Parallel studies with a rapidly growing EB-virus transformed lymphoblastoid line showed no hCG effect. In contrast to previous reports on the immunosuppressive action of hCG, we conclude that hCG functions, in our experimental conditions, as a mitogen and a stimulator of polyclonal immunoglobulin synthesis.     

Inhibition of human endothelial cell proliferation by gold compounds.

Neovascularization has a role in the propagation of rheumatoid synovitis because the spread of mononuclear cell infiltration and the growth of pannus are dependent on the growth of new blood vessels. Growth of such vessels requires local endothelial cell (EC) proliferation. Inhibition of synovial EC proliferation, therefore, would have the potential to diminish rheumatoid inflammation. We have, therefore, studied the effects of gold sodium thiomalate (GST), auranofin, and gold chloride on the proliferation of human umbilical vein EC. GST suppressed both basal and EC growth factor-induced tritiated thymidine incorporation into EC in a dose-dependent fashion. Inhibition was observed with concentrations as low as 1 microgram/ml GST, 5 micrograms/ml gold chloride, and 0.1 microgram/ml auranofin, levels attainable in blood and synovium of patients. These results suggest that gold compounds have an antiangiogenic effect. The low concentrations inhibiting EC proliferation suggest that gold compounds may suppress rheumatoid synovitis by reducing the number of small blood vessels available for mononuclear cell infiltration and synovial tissue proliferation.     

肿瘤抑素对婴幼儿血管瘤内皮细胞增殖活力与细胞周期的影响

目的探讨抗血管生成药物肿瘤抑素(tumstatin)对体外培养的婴幼儿血管瘤内皮细胞增殖与细胞周期的影响。方法将切取的血管瘤组织在体外消化,获得血管瘤内皮细胞,进行体外培养和传代;以不同浓度tumstatin多肽加入细胞培养基中,MTT法检测细胞活力,以流式细胞仪检测细胞周期的变化。结果在2.5%体积胎牛血清的培养条件下,tumstatin对血管瘤内皮细胞增殖活力的抑制,呈现出量效关系;在17.0μg/mL浓度tumstatin的作用下,血管瘤内皮细胞的细胞周期G0/G1期为55.8%,而对照组为42.9%。结论 tumstatin能够在体外有效地抑制婴幼儿血管瘤内皮细胞的增殖活力,并呈现出一定的量效关系;tumstatin还能影响细胞周期的进程,促使其阻滞在G0/G1期。     

Human lymphoblastoid cells produce extracellular matrix-degrading enzymes and induce endothelial cell proliferation, migration, morphogenesis, and angiogenesis

Human lymphoproliferative diseases can be hypothesized to invade locally and to metastatize via mechanisms similar to those developed by a variety of solid tumors, i.e., the secretion of extracellular matrix-degrading enzymes and stimulation of angiogenesis. To assess this hypothesis, Namalwa, Raji, and Daudi cell lines (Burkitt’s lymphoma), LIK and SB cell lines (B-cell lymphoblastic leukemia), CEM and Jurkat cell lines (T-cell lymphoblastic leukemia), and U266 cell line (multiple myeloma) were evaluated for their capacity to produce matrix metalloproteinase-2 and -9, and urokinase-type plasminogen activator. These cell lines were also assessed for their ability: (1) to produce the angiogenic basic fibroblast growth factor and vascular endothelial growth factor; (2) to induce an angiogenic phenotype in cultured endothelial cells, represented by cell proliferation, chemotaxis, and morphogensis; (3) to stimulate angiogenesis in different in vivo experimental models. All cell lines expressed the mRNA for one or both metalloproteinases. Namalwa, Raji, LIK, SB, and U266 cells secreted the active form of both metalloproteinases, while Daudi, CEM, and Jurkat cells produced metalloproteinase-2 but not -9. In contrast, urokinase-type plasminogen activator was secreted only by SB cells. While Raji, LIK, SB, CEM, and Jurkat cells secreted both basic fibroblast growth factor and vascular endothelial growth factor, Daudi and U266 cells produced only the former, and Namalwa cells only the latter. Accordingly, the conditioned medium of all cell lines stimulated cell proliferation and/or chemotaxis in cultured endothelial cells, with the exception of that of Namalwa cells which was ineffective. The conditioned medium of CEM and Jurkat cells induced morphogenesis in cultured endothelial cells grown on a reconstituted basement membrane (Matrigel). Lastly, Namalwa, Raji, LIK, SB, U266, CEM, and Jurkat cells induced angiogenesis and mononuclear cell recruitment in the murine Matrigel sponge model and in a chick embryo chorioallantoic membrane assay. The extent of angiogenesis in both models was strictly correlated with the density of the mononuclear cell infiltrate. The results indicate that human lymphoproliferative disease cells possess both local and remote invasive ability via the secretion of matrix-degrading enzymes and the induction of angiogenesis which is fostered by host inflammatory cells and by an intervening ensemble of angiogenic factors.     

Macrolides and immunity: effects of erythromycin and spiramycin on human mononuclear cell proliferation

Macrolides are actively concentrated by leucocytes. The dose-effect responses of spiramycin (Sp) and erythromycin (Er) on phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) stimulated human mononuclear leucocytes (MNL) were studied. Cell viability was not altered at any antibiotic concentration (1-100 mg/l). Both Sp and Er showed dose-related inhibition of the proliferative response of PHA and PWM stimulated MNL. Very marked effects were observed at high antibiotic concentrations and the effects observed at low concentrations (1-10), although small, were also significant. Similar results were observed for the mitogen PWM. A decrease in tritiated thymidine (3H-TdR) incorporation occurred only if Sp and Er were added during the first 8 h of culture. Sp and Er also induced a decrease in tritiated uridine (3H-UdR) uptake. These data suggest that Sp and Er interfered with an early event in the cell cycle. However Sp did not affect PHA binding to MNL. The clinical significance of these findings is discussed.     

Human mononuclear cell factors mediate cartilage matrix degradation through chondrocyte activation.

Human blood mononuclear cells (BMC) in short-term culture secrete one or more factors that induce degradation of matrix proteoglycan and collagen in cartilage explants in organ culture. Induction of matrix degradation took place both in nasal septum and articular cartilage explants in the presence of the mononuclear cell supernates. Cartilage degradation in this system was absolutely dependent on the presence of live chondrocytes. Matrix depletion did not occur in dead cartilage explants cultured with active supernates. Supernates obtained from unstimulated BMC showed variable cartilage matrix degrading activity (MDA). BMC stimulated with phytohemagglutinin (PHA) showed increased MDA, which in one dilution experiment was found to be five times higher than that in the unstimulated control supernate. Concanavalin A and pokeweed mitogen were also shown to stimulate release of MDA. Time experiments showed that most of the degrading activity was released by the mononuclear cells during the first day of culture. The cellular origin of MDA was investigated with the aid of partially purified BMC subpopulations. Removal of adherent cells resulted in a decrease of MDA release. Purified T lymphocytes failed to show enhanced MDA release in spite of their ability to mount a virtually intact proliferative response to PHA. Purified adherent cells also failed to show enhanced PHA-dependent MDA release. Nevertheless, restoration of PHA-dependent MDA release took place in reconstituted cell populations containing both T lymphocytes and monocytes. These experiments suggest that MDA may be released by adherent mononuclear cells, presumably monocytes, and that the PHA-dependent increase in MDA release may be mediated by T lymphocytes. Partial characterization of MDA by gel chromatography showed one active fraction corresponding to an apparent molecular weight ranging from 12,000 to 20,000. The fraction was also shown to degrade cartilage matrix only in the presence of live chondrocytes. These results demonstrate that factors released by human BMC mediate degradation of matrix proteoglycan and collagen in intact cartilage explants through chondrocyte activation. This pathogenic mechanism may play a role in in vivo cartilage destruction in chronic inflammatory joint diseases.     

内皮抑素重组腺病毒表达载体构建及对内皮细胞增殖的影响

背景:烧创伤后创面愈合多伴有瘢痕增生,内皮细胞在瘢痕增生中具有重要作用,抑制内皮细胞的生长可以在一定程度上减轻瘢痕增生。目的:构建含有人内皮抑素基因的重组腺病毒Ad/hEnd,并观察与感染该病毒的角朊细胞共培养时内皮细胞增殖特性的改变。设计、时间及地点:观察对照实验,于2006-09/2007-05在解放军第三军医大学西南医院烧伤研究所国家重点实验室完成。材料:pAdTrack-CMV及pAdEasy-1购自美国Stratagene公司;293细胞及大肠杆菌Ecoli.DH5α由本室保存。方法:以人胎肝组织mRNA为模板,通过反转录-聚合酶链反应及聚合酶链反应获得内皮抑素基因序列,插入到腺病毒穿梭质粒pAdTrack-CMV中,获得重组质粒pAdTrack-ES。经鉴定后将阳性重组子转化至pAdeasy1受体菌,筛选阳性克隆。脂质体介导转染293细胞,获取Ad/hEnd,经聚合酶链反应鉴定及上清中内皮抑素含量检测后,感染角朊细胞,采用套皿法与内皮细胞共培养。并与未转染的角朊细胞进行对照观察。主要观察指标:pAd/hEnd的同源重组及其鉴定,Ad/hEnd的产生与鉴定,转染293细胞后内皮抑素的表达,Ad/hEnd的纯化与滴度测定,培养液中内皮抑素含量,内皮细胞凋亡百分数及内皮细胞抑制率测定。结果:①成功获取了Ad/hEnd,病毒滴度可达1.65×1012PFU/L。②感染Ad/hEnd的角朊细胞可有效表达并分泌内皮抑素,连续培养3d后,培养液中内皮抑素含量可达226μg/L。③与转基因角朊细胞共培养的内皮细胞凋亡百分数与细胞抑制率均显著高于对照组(P<0.05)。结论:与内皮细胞共培养时,转Ad/hEnd角朊细胞可通过分泌内皮抑素促进内皮细胞凋亡,并抑制其增殖。     

Identification of biomarkers for tumor endothelial cell proliferation through gene expression profiling

Extensive efforts are under way to identify antiangiogenic therapies for the treatment of human cancers. Many proposed therapeutics target vascular endothelial growth factor (VEGF) or the kinase insert domain receptor (KDR/VEGF receptor-2/FLK-1), the mitogenic VEGF receptor tyrosine kinase expressed by endothelial cells. Inhibition of KDR catalytic activity blocks tumor neoangiogenesis, reduces vascular permeability, and, in animal models, inhibits tumor growth and metastasis. Using a gene expression profiling strategy in rat tumor models, we identified a set of six genes that are selectively overexpressed in tumor endothelial cells relative to tumor cells and whose pattern of expression correlates with the rate of tumor endothelial cell proliferation. In addition to being potential targets for antiangiogenesis tumor therapy, the expression patterns of these genes or their protein products may aid the development of pharmacodynamic assays for small molecule inhibitors of the KDR kinase in human tumors.     

Activation of vascular endothelial growth factor through reactive oxygen species mediates 20-hydroxyeicosatetraenoic acid-induced endothelial cell proliferation

20-Hydroxyeicosatetraenoic acid (20-HETE) is formed by the omega-hydroxylation of arachidonic acid by cytochrome P450 4A and 4F enzymes, and it induces angiogenic responses in vivo. To test the hypothesis that 20-HETE increases endothelial cell (EC) proliferation via vascular endothelial growth factor (VEGF), we studied the effects of WIT003 [20-hydroxyeicosa-5(Z),14(Z)-dienoic acid], a 20-HETE analog on human macrovascular or microvascular EC. WIT003, as well as pure 20-HETE, stimulated EC proliferation by approximately 40%. These proliferative effects were accompanied by increased VEGF expression and release that were observed as early as 4 h after 20-HETE agonist addition. This was accompanied by increased phosphorylation of the VEGF receptor 2. The proliferative effects of 20-HETE were markedly inhibited by a VEGF-neutralizing antibody. Polyethylene glycol-superoxide dismutase (PEG-SOD) markedly inhibited both the increases in VEGF expression and the proliferative effects of 20-HETE. In contrast, administration of the NAD(P)H oxidase inhibitor apocynin had no effect to the proliferative response to 20-HETE. The 20-HETE agonist markedly increased superoxide formation as reflected by an increase in dihydroethidium staining of EC, and this increase was inhibited by PEG-SOD but not by apocynin. 20-HETE also increased the phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK) in EC, whereas an inhibitor of MAPK [U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] suppressed the proliferative and the VEGF changes but not the pro-oxidant effects of 20-HETE. These data suggest that 20-HETE stimulates superoxide formation by pathways other than apocynin-sensitive NAD(P)H oxidase, thereby activating MAPK and then enhancing VEGF synthesis that drives EC proliferation. Thus, 20-HETE may be involved in the regulation of EC functions, such as angiogenesis.     

CXCL4L1 inhibits angiogenesis and induces undirected endothelial cell migration without affecting endothelial cell proliferation and monocyte recruitment

Summary. Background and Objectives: The non‐allelic variant of CXCL4/PF4, CXCL4L1/PF4alt, differs from CXCL4 in three amino acids of the C‐terminal α‐helix and has been characterized as a potent anti‐angiogenic regulator. Although CXCL4 structurally belongs to the chemokine family, it does not behave like a ‘classical’ chemokine, lacking significant chemotactic properties. Specific hallmarks are its angiostatic, anti‐proliferative activities, and proinflammatory functions, which can be conferred by heteromer‐formation with CCL5/RANTES enhancing monocyte recruitment. Methods and Results: Here we show that tube formation of endothelial cells was inhibited by CXCL4L1 and CXCL4, while only CXCL4L1 triggered chemokinesis of endothelial cells. The chemotactic response towards VEGF and bFGF was attenuated by both variants and CXCL4L1‐induced chemokinesis was blocked by bFGF or VEGF. Endothelial cell proliferation was inhibited by CXCL4 (IC₅₀ 6.9 μg mL^?1) but not by CXCL4L1, while both chemokines bound directly to VEGF and bFGF. Moreover, CXCL4 enhanced CCL5‐induced monocyte arrest in flow adhesion experiments and monocyte recruitment into the mouse peritoneal cavity in vivo, whereas CXCL4L1 had no effect. CXCL4L1 revealed lower affinity to CCL5 than CXCL4, as quantified by isothermal fluorescence titration. As evidenced by the reduction of the activated partial thromboplastin time, CXCL4L1 showed a tendency towards less heparin‐neutralizing activity than CXCL4 (IC₅₀ 2.45 vs 0.98 μg mL^?1). Conclusions: CXCL4L1 may act angiostatically by causing random endothelial cell locomotion, disturbing directed migration towards angiogenic chemokines, serving as a homeostatic chemokine with a moderate structural distinction yet different functional profile from CXCL4.     

没食子儿茶素没食子酸酯抑制牛主动脉内皮细胞增殖和对细胞周期分布的影响

背景:血管内皮细胞在肿瘤血管生成中起关键作用,没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)可抑制内皮细胞生长和增殖,但其作用机制仍不清楚.目的:观察没食子儿茶素没食子酸酯对牛主动脉内皮细胞增殖和细胞周期分布的影响.设计、时间及地点:以细胞为观察对象的随机分组实验,于2006-08/2008-05在广州医学院完成.材料:出生1 d、未哺乳的新生牛,取主动脉内皮细胞进行原代培养.方法:将指数生长期密度为5×107L-1的牛主动脉内皮细胞接种于96孔培养板,实验分为2组:实验组和对照组分别加入以培养基RPMI 1640配制的不同浓度的EGCG 180 μL(终浓度为5,10,20,40,80 g/L)及等体积不含EGCG的培养基,全自动酶标仪上测定不同时间3,6,12,24,36 h各孔吸光度值,并计算细胞生长抑制率.选择适当的药物浓度和作用时间点进行EGCG抑制作用的半数抑制浓度(IC50)实验.收集对数牛长期的牛主动脉内皮细胞,实验组加入不同浓度(10,20,30,40 g/L)的EGCG,对照组加入不含药物的培养基,Multicycle软什分析G1/G0期、S期和G2/M期细胞所占比例.主要观察指标:①不同剂量EGCG在不同时间点对牛主动脉内皮细胞生长、增殖的影响.②运用直线回归分析EGCG对牛主动脉内皮细胞抑制的IC50.③流式细胞仪检测EGCG对内皮细胞周期分布的影响.结果:①EGCG呈剂量及时间依赖性抑制内皮细胞的增殖:EGCG作用12 h后,细胞抑制率达高峰,此后其抑制率逐渐下降(P<0.05);从10 g/L起,EGCG对内皮细胞增殖的抑制效应逐渐增加,40 g/L即达高峰(P<0.05);40 g/L与80 g/L剂量组相比,抑制率无明显差异(P>0.05).②在3,6,12,24 h和36 h等时间点EGCG的IC<50分别为23.14,18.79,13.23,14.19,17.45 g/L,IC50在12 h达高峰,时间继续延长,作用并不增加.③各组G2/M期细胞所占比率无明显差异(P>0.05).S期细胞比率由(58.02±1.21)%下降为(27.62±3.12)%,其中30 g/L和40 g/L剂量组与对照组相比,差异有显著性意义(P<0.01).G0/G1期细胞比率由(29.22±3.21)%上升到(62.32±4.11)%,其中30 g/L和40 g/L剂量组与对照组相比,差异有显看性意义(P<0.01).结论:EGCG在体外能有效抑制牛主动脉内皮细胞增殖,且使其周期主要受阻于G0/G1期,可能是其抑制内皮细胞增殖的机制之一.     

Homocysteine-induced modulation of tissue plasminogen activator binding to its endothelial cell membrane receptor.

Endothelial cells impart thromboresistance to the blood vessel wall. As modulators of fibrinolytic activity, these cells synthesize and secrete tissue plasminogen activator (t-PA) as well as its physiologic inhibitor, plasminogen activator inhibitor-1. In addition, endothelial cells support membrane-associated assembly of plasminogen and tissue plasminogen activator. Recently, an M(r) approximately 40,000 protein expressed on endothelial cells has been shown to interact noncompetitively through disparate mechanisms with both t-PA and plasminogen, suggesting trimolecular assembly of enzyme, substrate, and receptor (Hajjar, K. A. 1991. J. Biol. Chem. 266:21962-21970). In the present study, treatment of cultured endothelial cells with DL-homocysteine was specifically associated with a selective reduction in cellular binding sites for t-PA. This 65% decrease in binding was associated with a 60% decrease in cell-associated t-PA activity. No change in affinity for t-PA or plasminogen or in the maximal number of binding sites for plasminogen was observed. Matrix-associated t-PA binding sites were not affected. These data suggest a new mechanism whereby homocysteine may perturb endothelial cell function, thus promoting a prothrombotic state at the surface of the blood vessel wall.     

碱性成纤维细胞生长因子促进猫角膜内皮细胞的增殖作用

背景:有实验报道,碱性成纤维细胞生长因子能改变角膜内皮细胞形状、促进其分裂和趋化,刺激活体和体外角膜内皮细胞的损伤修复.目的:观察碱件成纤维细胞生长因子对猫角膜内皮细胞增殖的影响.设计、时间及地点:细胞肜态学观察实验,2005-01/2006-12在青岛大学医学院附属医院中心实验室完成.材料:家猫购买自青岛大学医学院附属医院动物实验中心.方法:猫角膜内皮细胞原代培养,行NSE免疫组织化学染色鉴定细胞的类型、纯度及生理特性.传1代后,分别在培养液中加1,10,100 μ L的碱性成纤维细胞生长因子,继续卵化1～5 d.主要观察指标:加药后第1,3,5天分别利用MTT法测定在490 nm处的吸光度值来判断角膜内皮细胞增殖情况;同时应用倒置相筹显微镜观察细胞形态及透射电镜检测细胞超微结构.结果:碱性成纤维细胞生长因子作用组加药后1,3,5 d,MTT法测定猫角膜内皮细胞在490 nm处的吸光度值明显高于对照组,其中10 μ g/L作用组差别最显著.结论:碱性成纤维细胞生长因子作用能促进猫角膜内皮细胞的增殖,相对有效浓度为10 μ g/L.     

外加直流电场对内皮细胞增殖与迁徙的影响

背景:已有研究证明,电刺激可以引起内皮细胞的增殖、趋阴迁徙,同时伴随细胞分泌物释放的增加,从而调节血管张力和血管平滑肌细胞的生长,介导血管收缩和舒张.目的:观察外加直流电场对人脐静脉内皮细胞HY926增殖和迁徙的影响.方法:MTT法检测在外加电压3,5, 8,10 V作用12 h后人脐静脉内皮HY926细胞的增殖情况.并选择内皮细胞增殖最佳的外加电压5 V,在5 V外加电压作用10 h内追踪人脐静脉内皮HY926细胞的运动轨迹,包括细胞的平均累计移行距离和平均移行速率,细胞的平均最终位移大小及其方向,以及所有单个细胞每小时的平均追踪sine值.结果与结论:在外加电压3,5,8,10 V下作用12 h,人脐静脉内皮细胞生长均受到不同程度的抑制,但在外加电压5 V,电场强度163.46 mV/cm时内皮细胞增殖情况最好.在该生理电场下培养72 h,发现细胞生长贴壁生长、增殖正常,初步说明该生物反应器的细胞相容性良好,并且随时间的延长,内皮细胞的平均移行速率呈下降趋势.在5 V外加电压作用10 h后,内皮细胞整体明显持续朝向阴极方向迁徙.提示外加直流电场对人脐静脉内皮细胞增殖有显著的抑制作用,并且可诱导细胞定向向阴极迁徙.     

Proliferative glomerulonephritis in rats: evidence that mononuclear phagocytes infiltrating the glomeruli stimulate the proliferation of endothelial and mesangial cells

Abstract. A proliferative, non-crescentic, glomerulonephritis (GN) was induced in rats preimmunized with rabbit IgG by injecting a sub-nephrotoxic dose of rabbit anti-GBM IgG. Control rats either received anti-GBM IgG only, or were totally irradiated (800 rads, kidneys protected) 2 days before the second injection. All the GN rats developed a severe proteinuria within 2–4 days after the injection of anti-GBM IgG, contrarily to the control rats. At the same time, many mononuclear cells, of predominantly extra-renal origin, infiltrated the glomeruli. Glomeruli were isolated from GN, normal and control rats and were cultivated in RPMI medium. In normal and control rat cultures, epithelial and mesangial cells were observed. In GN rat cultures, not only epithelial and mesangial cells, but also endothelial and macrophagic cells were identified; the outgrowth capacity of the mesangial cells was enhanced. These data were particularly evident in cultures of GN glomeruli isolated within 2–4 days after the induction of the renal disease, exactly when the glomeruli were infiltrated by a large number of mononuclear phagocytes. It is suggested that the mononuclear phagocytes infiltrating the glomeruli of rats with this model of GN stimulate the proliferation of endothelial and mesangial cells in vitro.     

血管内皮生长因子联合骨髓单个核细胞自体移植治疗大鼠肢体缺血

目的:将血管内皮生长因子应用于骨髓单个核细胞自体移植治疗肢体缺血,探索一种简单、安全有效治疗肢体缺血的方法,同时初步分析血管内皮生长因子促进血管新生的机制和疗效。方法:实验于2005-03/2006-07在上海交通大学附属第六人民医院动物实验中心完成。将48只雄性Wistar大鼠采用随机单位组设计分为4组:缺血对照组、血管内皮生长因子治疗组、骨髓单个核细胞治疗组、转染血管内皮生长因子基因的骨髓单个核细胞治疗组,每组12只。结扎所有动物左后肢股动脉及分支,制备后肢缺血模型。以微量加样器自结扎处以下2cm开始,间隔0.2cm为一注射点,共注射于内收肌和腓肠肌5点,每注射点各注射60μL,缺血对照组大鼠注射300μL培养液,血管内皮生长因子治疗组大鼠注射含5μg基因pcDNA3.1-hVEGF165的DNA-脂质体复合物,骨髓单个核细胞治疗组大鼠注射3×106个骨髓单个核细胞,转染血管内皮生长因子基因的骨髓单个核细胞治疗组大鼠注射含3×106个血管内皮生长因子的骨髓单个核细胞。于移植后4周行动脉造影,采用RT-PCR检测血管内皮生长因子基因的体内表达,采用免疫组化法检测缺血区新生血管密度,评价血管新生情况。结果:48只大鼠均造模成功,并进入结果分析。①移植后4周动脉造影显示,缺血对照组大鼠股动脉及其分支均未显影,血管内皮生长因子治疗组和骨髓单个核细胞治疗组可见中等量新生血管,两组效果相似,转染血管内皮生长因子基因的骨髓单个核细胞治疗组可见有大量新生血管新生,丰富的侧枝循环建立。②大鼠毛细血管密度测定结果显示,血管内皮生长因子治疗组和骨髓单个核细胞治疗组均较缺血对照组新生血管数量增加,转染血管内皮生长因子基因的骨髓单个核细胞治疗组可进一步增强疗效[(10.0±0.8),(21.7±1.9),(20.4±3.3),(42.1±3.5)个/HP,P<0.05],血管内皮生长因子治疗组和骨髓单个核细胞治疗组两组之间差异无明显统计学意义(P>0.05)。③RT-PCR检测大鼠血管内皮生长因子基因的体内表达,转染血管内皮生长因子基因的骨髓单个核细胞治疗组和血管内皮生长因子治疗组均有扩增,转染血管内皮生长因子基因的骨髓单个核细胞治疗组人血管内皮生长因子mRNA相对含量表达高于血管内皮生长因子治疗组(0.191±0.044,0.094±0.032,P<0.05)。结论:本组实验结果说明采用骨髓单个核细胞作为基因载体细胞治疗大鼠肢体缺血,能使血管内皮生长因子获得稳定有效的表达,其效果优于直接基因注射。