低氧刺激诱导体外培养的大鼠脑皮质星形胶质细胞内neuregulin-1表达上调

Neuregulin-1(NRG-1)是神经胶质及神经元产生的细胞间信号转导蛋白。该蛋白通过与ErbB受体结合,对神经系统的正常发育、成熟发挥重要作用,也在缺血性脑损伤时起神经保护作用。为探讨体外培养的星形胶质细胞受缺氧刺激后NRG-1的表达特点,本实验利用体外培养的大鼠脑皮质星形胶质细胞,采用免疫组织化学和Westernblot方法,比较了正常培养和低氧复氧条件下星形胶质细胞中NRG1的表达。结果显示:正常培养的星形胶质细胞中有NRG-1的表达,但含量不高;低氧复氧培养后星形胶质细胞NRG1的表达量随着复氧时间的延长缓慢增加,至复氧8h,其表达量陡然升高,达到峰值后又逐渐降低,甚至降至正常水平以下。本研究表明,在低氧缺血性脑损伤后,星形胶质细胞反应性大量产生NRG1的时间相对滞后。由此提示,低氧缺血性脑损伤后,立即使用外源性神经保护剂为宜。     

细胞外调节蛋白激酶在体外培养下大鼠大脑皮质星形胶质细胞的表达

目的:研究大鼠大脑皮质星形胶质细胞细胞外调节蛋白激酶(ERK)表达.方法:用生后2～3d SD大鼠,在无菌条件下取大脑皮质,制备细胞悬液,以GFAP鉴定星形胶质细胞;用免疫细胞化学方法研究星形胶质细胞ERK1表达.结果:星形胶质细胞的纯度达95%以上,ERK1免疫阳性物质呈棕黄色,分布于星形胶质细胞胞体与突起中.结论:培养的大鼠星形胶质细胞表达ERK1,其可能与星形胶质细胞信号转导有关.     

大鼠脑皮质星形胶质细胞体外培养方法的改进

目的改进大鼠脑皮质星形胶质细胞体外培养方法,获得纯度较高的大鼠脑皮质星形胶质细胞。方法取出生后1d内SD大鼠大脑皮质,用机械吹打法使细胞分散制成初细胞悬液,10μm尼龙膜过滤除去成纤维细胞后接种。待7d细胞铺满瓶底后,＂十＂字形震荡培养液5min后传代接种。传代2次后行GFAP免疫组化染色鉴定。结果通过微孔尼龙膜过滤和传代前＂十＂字形震荡培养液等操作可以得到形态典型、纯度较高的星形胶质细胞。通过纯度计算可得离体培养的星形胶质细胞的纯度平均值为95.49%（n=6）。结论改进的星形胶质细胞体外培养方法,降低了实验操作难度,方法稳定有效,得到的星形胶质细胞纯度较高。     

活化小胶质细胞致星形胶质细胞激活

目的探讨活化小胶质细胞培养液对星形胶质细胞的影响。方法 LPS激活原代培养小胶质细胞,采用活化的小胶质细胞条件培养液刺激星形胶质细胞,观察星形胶质细胞GFAP及IL-1β和TNFα的表达。结果 LPS刺激后,小胶质细胞OX42表达量上升,IL-1β和TNFα的表达量增高;小胶质细胞条件培养液可致星形胶质细胞激活,GFAP表达量上升,IL-1β和TNFα的表达量增加。结论活化小胶质细胞的条件培养液可致星形胶质细胞激活,激活的小胶质细胞和星形胶质细胞表达前炎症介质IL-1β和TNFα。     

氯喹抑制体外培养星形胶质细胞的激活

目的研究氯喹对体外培养大鼠海马星形胶质细胞激活的抑制作用,为癫痫的治疗提供实验依据。方法分离新生SD大鼠海马,体外培养星形胶质细胞,经纯化鉴定后分为:对照组、戊四氮(PTZ)组、氯喹干预组(25、50和75 mg/L),经相应处理后,分别用MTT法、免疫荧光、Western blot测定星形胶质细胞数量及活性、星形胶质纤维酸性蛋白(GFAP)及CyclinD1的表达量。结果与对照组比较,PTZ可激活星形胶质细胞的增殖,使GFAP、CyclinD1的表达量增加(P<0.05);与PTZ组比较,氯喹阻滞了PTZ激活的星形胶质细胞的增殖(P<0.05);氯喹可抑制PTZ激活星形胶质细胞异常增加的GFAP的表达;氯喹可抑制PTZ激活星形胶质细胞的CyclinD1的表达量(P<0.05);与对照组相比,3种结果均显示75 mg/L氯喹对体外培养星形胶质细胞激活的抑制作用较强,并可维持其在正常范围。结论氯喹具有抑制PTZ激活体外培养星形胶质细胞的作用,其可能通过抑制星形胶质细胞的增殖来发挥抗癫痫作用。     

芍药甙对体外培养大鼠星形胶质细胞活性的作用

目的　观察中药芍药有效成分芍药甙 (paeoniflorin ,PF)对培养的大脑皮层星形胶质细胞活性的影响。方法　采用体外星形胶质细胞培养的方法 ,对新生SD大鼠 (P2 )大脑星形胶质细胞进行培养 ,7d后纯化 ,再分别进行有血清和无血清培养。以MTT法观察PF对星形胶质细胞活性的影响 ,运用免疫组织化学的方法进行形态学观察。　结果　PF对星形胶质细胞活性有明显的抑制作用 ,不同浓度PF组星形胶质细胞活性均低于对照组 (P <0 0 1) ,并呈现出一定的量效关系 ,这种对星形胶质细胞活性的抑制作用与血清的存在与否无关。　结论　本实验表明芍药甙能抑制星形胶质细胞的活性 ,从而提示PF促进神经细胞活性的作用是一种直接的作用 ,而不是通过胶质细胞间接影响神经细胞     

体外炎性诱导星形胶质细胞激活及功能分析

目的 观察体外培养的星形胶质细胞在炎性刺激下的激活及功能变化。方法 体外培养C57BL/6小鼠皮层的星形胶质细胞,应用免疫细胞化学和Western blot等方法,测定炎性刺激下各组细胞形态学及促炎因子（iNOS、IL-6、TNF-α）和抗炎因子（ARG1、IL-10、TGF-β1）表达水平的变化情况。结果炎性刺激后,免疫荧光细胞染色结果显示,M1(LPS+IFN-γ)组细胞被异常激活,胞体出现肿胀变大、扭曲等形态改变,且GFAP阳性细胞数量明显增多（P<0.001 vs. control））。CCK-8细胞活力检测结果显示,LPS组、IFN-γ组及M1组的吸光度均升高,其中M1组显著升高（P<0.001 vs. control）。Western blot结果表明,M1组促炎因子（iNOS、IL-6和TNF-α）表达均明显升高（P<0.001 vs. control）;各组抗炎因子（ARG1、IL-10和TGF-β1）的表达也显著升高（P<0.01 or 0.001 vs. control）。结论 体外炎性刺激异常激活星形胶质细胞,形态和功能显著变化,且A1样星...     

血管内皮细胞生长因子促进成年小鼠胸腺细胞的增殖与分化

目的 研究血管内皮生长因子（VEGF121）在免疫系统的表达及其对小鼠胸腺细胞的作用。为了解VEGF在免疫系统中的功能提供实验依据。方法 构建pCDI-VEGF真核表达载体，转染COS-7细胞，收获上清检测其对小鼠胸腺细胞的促增殖效应和CD4，CD8分子表达，用抗VEGF单克隆抗体及不同信号转导抑制剂进行阻断实验。通过RT-PCR方法检测人VEGFmRNA在胸腺，脾脏等免疫器官的表达。结果 实验证明VEGFmRNA可以在多种免疫组织中表达。VEGF121真核表达蛋白可明显促进小鼠胸腺细胞增殖和分化。此作用可被VEGF的单克隆抗体所封闭；不同的信号转导抑制剂具有不同的效应。结论 VEGF参与了免疫系统的功能，对小鼠胸腺细胞的增殖分化有促进作用。可能是内源性的免疫系统的调节因子。     

Effects of high-altitude exposure on vascular endothelial growth factor levels in man

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen and permeability factor that is inducible by hypoxia. Its contribution to high-altitude illness in man is unknown. We measured VEGF levels in 14 mountaineers at low altitude (490 m) and 24 h after their arrival at high altitude (4,559 m). At high altitude, VEGF increased from [mean (SEM)] 32.5 (9.2) to 60.9 (18.5) pg·ml^–1 (P<0.004) in the arterial blood, and from 15.9 (2.9) to 49.3 (15.9) pg·ml^–1 (P=0.0001) in the mixed venous blood. Whereas at low altitude venous and arterial VEGF levels were not statistically different from each other (P=0.065), the VEGF concentration was significantly lower in venous than in arterial blood samples at high altitude (P=0.004). The pulmonary capillary VEGF concentration remained unchanged at high altitude [14.8 (2.5) vs 17.1 (5.4) pg·ml^–1, P=0.85]. VEGF levels in the nine mountaineers who developed symptoms of acute mountain sickness (AMS), and in the six subjects who had radiographic evidence of high-altitude pulmonary edema were similar to those in subjects without symptoms. VEGF was not correlated with either AMS scores, mean pulmonary arterial pressures, arterial partial pressure of O₂, or alveolar-arterial O₂ gradients. We conclude that VEGF release is stimulated at high altitude, but that VEGF is probably not related to high-altitude illness. Electronic Publication     

Hypoxia and cobalt stimulate vascular endothelial growth factor receptor gene expression in rats

 This study aimed to examine the influence of acute tissue hypo-oxygenation on the expression of the vascular endothelial growth factor (VEGF) receptor genes. To this end male Sprague-Dawley rats were exposed to different hypoxic conditions such as 10% or 8% oxygen, 0.1% carbon monoxide and cobalt chloride (60 mg/kg) for 6 h and the abundance of flt-1, flt-4 and flk-1 mRNA in lungs and livers was determined by RNase protection assay. The relative proportions of flt-1, flt-4 and flk-1 were 10 : 2.5 : 1 and 10 : 10 : 2 in normoxic lungs and livers, respectively. It was found that 8% but not 10% oxygen increased flt-1 mRNA two- to threefold in both organs, whilst flt-4 and flk-1 mRNA were not changed by acute inspiratory hypoxia. Carbon monoxide inhalation also increased flt-1 mRNA but not flt-4 or flk-1 mRNA in both organs. Subcutaneous cobalt administration increased flt-1 mRNA in the livers only, whilst flt-4 and flk-1 mRNA remained unchanged. These findings show that acute tissue hypo-oxygenation is a rather selective stimulus for flt-1 gene expression. The efficiency of the different manoeuvres applied to stimulate flt-1 gene expression is rather similar to the stimulation of erythropoietin gene expression. It is not unreasonable to assume, therefore, that the oxygen-dependent regulation of both genes at the cellular level has significant similarities.     

Hypoxia-induced expression of vascular endothelial growth factor by retinal glial cells promotes in vitro angiogenesis

To determine whether retinal glial cells (RGCs) participate in the paracrine regulation of retinal neovascularization, we investigated whether cultured RGCs synthesize and release vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) under normoxic or hypoxic conditions. Northern blot analysis demonstrated that cultured RGCs transcribed both VEGF mRNA with two molecular bands approximately 3.9 and 4.3 kilobases (kb), and bFGF mRNA with approximately 3.7 and 6.0 kb. The expression of VEGF mRNA was greatly enhanced by hypoxic cultivation (2% oxygen) when compared with normoxic cultivation (20% oxygen), while the expression of bFGF mRNA by RGCs was not significantly affected by hypoxia. The effects of RGCs-conditioned media (CM) on tritiated-thymidine incorporation and in vitro angiogenesis by retinal capillary endothelial cells (RECs) in producing the formation of capillary-like tubes in type I collagen gels, were evident in the observation that RGCs-CM harvested after hypoxic cultivation significantly enhanced tritiated-thymidine incorporation (1.9 times, P<0.01) and in vitro angiogenesis (2.4 times, P<0.01) compared with the normoxic RGCs-CM. These enhancing effects of RGCs-CM at hypoxia were suppressed by anti-VEGF neutralizing antibody. Furthermore, RECs were shown to express mRNA encoding the VEGF receptor flt-1 by northern blot analysis. These results suggest that VEGF expressed by RGCs under hypoxic conditions plays an integral role in the initiation and progression of retinal neovascularization in a paracrine manner.     

Effects of vascular endothelial growth factor released from cultured dermal substitute on proliferation of vascular endothelial cells in vitro

Allogeneic cultured dermal substitute (CDS) was prepared by culturing fibroblasts on a two-layered spongy matrix of hyaluronic acid and atelocollagen. CDS can be cryopreserved and transported to other hospitals in a frozen state. The present study was designed to analyze the amounts of vascular endothelial growth factor (VEGF) released from fibroblasts in fresh and cryopreserved CDS and to investigate the effects of this VEGF on proliferation of vascular endothelial cells in vitro. The culture medium used in preparing CDS (fresh CDS culture medium sample) was collected and stored at –30°C for the quantitative analysis of VEGF. After thawing cryopreserved CDS, it was recultured in a culture medium for 1 week. The culture medium used was collected and stored at –30°C for quantitative analysis of VEGF. The amounts of VEGF released from the fresh and cryopreserved CDS into the culture medium were about 610pg/ml and 640pg/ml, respectively. This finding suggests that the cryopreserved CDS retains its ability to release VEGF. Immunohistological analysis indicated that some of the VEGF adhered to the matrix. Human vascular endothelial cells were cultured in medium mixed with the fresh or cryopreserved CDS culture medium sample. Proliferation of vascular endothelial cells was enhanced by increasing the concentration of both CDS culture medium samples. When antihuman VEGF antibody was added to the culture medium, the proliferative activity of vascular endothelial cells was reduced. These findings confirm that VEGF released from CDS promotes proliferation of vascular endothelial cells.     

血管内皮生长因子的免疫学测定法

目的：检测肿瘤细胞条件培养液中ＶＥＧＦ的表达量。方法：在表达及分离纯化ＶＥＧＦ的基础上,成功地制备了ＶＥＧＦ抗体,以夹心法测定ＶＥＧＦ,ＶＥＧＦ测定范围为０．０２～２００．００ｎｇ／ｍｌ,灵敏度可达０．０２ｎｇ／ｍｌ,批内及批间变异均小于１０％,回收率平均为１０２．４％。其中人胃癌细胞ＭＧＣ８０３、ＢＧＣ８２３、乳癌ＮＭＣＦ－７及肝癌ＢＥＬ－７４０２分泌上清中ＶＥＧＦ的含量分别为６．５０、０．４５     

Vascular endothelial growth factor expression in rat skin incision wound

Vascular endothelial growth factor (VEGF) is a glycoprotein that enhances vascular permeability, induces chemotaxis and activation of monocytes/macrophages, and promotes growth of vascular endothelial cells. We report that infiltrating polymorphonuclear leukocytes in an incision wound in rat skin express VEGF from 1 day after the injury, as shown by immunohistochemistry. VEGF is also present in macrophages, fibroblast-like cells, and endothelial cells 3 and 7 days after the injury. mRNA for VEGF is statistically significantly increased in the wound area in the tissue 1 day after the skin incision compared with 3 and 7 days after the incision. The VEGF protein content in the wound tissue is statistically significantly higher in the wound than in control skin at 1 and 3 days after skin incision. Our results indicate that VEGF is produced by inflammatory cells to induce vascularization in the early stage of the wound healing process.     

Divergent regulation of vascular endothelial growth factor and of erythropoietin gene expression in vivo

There is accumulating evidence from in vitro experiments that the gene expression of the vascular endothelial growth factor (VEGF) is, like that of the erythropoietin (EPO) gene, regulated by the oxygen tension and by divalent cations such as cobalt. Since the information about the regulation of VEGF gene expression in vivo is rather scarce, this study aimed to examine the influence of hypoxia and of cobalt on VEGF gene expression in different rat organs and to compare it with that on EPO gene expression. To this end male Sprague-Dawley rats were exposed to carbon monoxide (0.1% CO), hypoxia (8% O₂ ) or to cobalt chloride (12 and 60 mg/kg s.c.) for 6 h. mRNA levels for VEGF- 188, -164, and -120 amino acid isoforms in lungs, hearts, kidneys and livers were semiquantitated by RNase protection. For these organs we found a rank order of VEGF mRNA abundance of lung >> heart > kidney = liver. EPO mRNA levels were semiquantitated in kidneys and livers. Hypoxia, CO and cobalt increased EPO mRNA levels 60-fold, 140-fold and 5-fold, respectively, in the kidneys, and 11-fold, 11-fold and 3-fold, respectively, in the livers. None of these manoeuvres caused significant changes of VEGF mRNA in lung, heart or kidneys. Only in the livers did hypoxia lead to a significant (50%) increase of VEGF mRNA. These findings suggest that, in contrast to the in vitro situation, the expression of the VEGF gene in normal rat tissues is rather insensitive to hypoxia. In consequence, the in vivo regulation of the VEGF and the EPO genes appear to differ substantially, suggesting that the regulation of the VEGF and EPO genes may not follow the same essential mechanisms in vivo. Received: 31 July 1995/Received after revision: 20 November 1995/Accepted: 27 November 1995     

The effect of epidermal growth factor on vascular endothelial growth factor secretion by endometrial stromal cells

Endometrial stromal cells undergo morphological and functional changes to facilitate oocyte implantation under regulation of various hormones and growth factors. We studied physiological induction by epidermal growth factor (EGF) of vascular endothelial growth factor (VEGF) in these cells. In human endometrial stromal cells, the effect of EGF, genistein, tryphostin AG1478 (a tyrosine kinase inhibitor), and wortmannin (a phosphatidylinositol 3-kinase inhibitor) on production of VEGF was examined: Total RNA was extracted and VEGF mRNA expression was quantified by Northern analysis. EGF induced production of VEGF by stromal cells in a time-dependent manner; the effect became significant after 12 h and increased further between 24 and 48 h (P<0.05). Dose dependency was also significant (P<0.01). Genistein, tryphostin AG1478, and wortmannin partially suppressed the increase in production induced by EGF (P<0.01, P< 0.01, P<0.01), respectively. Production of EGF by fertilized oocytes and trophoblasts has been reported in early pregnancy. VEGF is believed to be induced by EGF through mechanisms involving tyrosine kinase and phosphatidylinositol 3-kinase. The increase in VEGF may contribute to neovascularization that promotes proliferation of endometrium and placentation. Received: 6 September 2001 / Accepted: 21 May 2002