血管活性肠肽对支气管上皮细胞趋化迁移的影响及机制

为探讨肺内神经肽在气道损伤修复中的作用 ,采用blind wellBoydenchamber测定原代培养的支气管上皮细胞 (bronchialepithelialcells,BEC)趋化性 ,观察血管活性肠肽 (vasoactiveintestinalpeptide ,VIP)对BEC趋化迁移的影响及其机制 ,并测定经热应激后BEC分泌VIP及表达VIP受体 (vasoactiveintestinalpeptidereceptor,VIPR)的变化。结果显示 :(1)以胰岛素作为趋化因子所建立的BEC趋化性测定方法稳定 ,重现性好 (r =0 970 3,P <0 0 1) ;(2 )VIP (0 0 0 1～ 1μmol/L)均显示剂量依赖性地增强BEC的趋化迁移 ,其效应可被钙调蛋白阻断剂及蛋白激酶C阻断剂有效地抑制 (P <0 0 1) ;(3) 4 2℃、30min热应激后BEC分泌VIP (P <0 0 1)及表达VIPR明显增加 (P <0 0 5 )。实验表明 :肺内神经肽VIP可增强BEC的趋化迁移 ,其细胞内信号转导途径与钙调蛋白及蛋白激酶C有关。而热应激时VIP及VIPR的高表达进一步提示局部微环境的VIP可能是气道上皮损伤修复网络中的重要分子     

血管活性肠肽对兔支气管上皮细胞抗臭氧损伤的保护作用

用支气管刷洗法收集新西兰兔支气管上皮细胞（ＢＥＣ）,以臭氧（Ｏ３）攻击培养的ＢＥＣ,建立细胞损伤模型。测定ＢＥＣ的３Ｈ释放率计算Ｏ３的细胞毒指数（ＣＩ）、测定细胞内丙二醛（ＭＤＡ）的含量反映细胞氧化性损伤的程度,测定细胞内过氧化氢酶（ＣＡＴ）活性及还原型和氧化型谷胱甘肽（ＧＳＨ和ＧＳＳＧ）的含量反映细胞抗氧化能力。观察血管活性肠肽（ＶＩＰ）预处理对ＢＥＣ的细胞保护作用并初步探讨其保护机制。观察到：ＢＥＣ的３Ｈ释放率与Ｏ３暴露时间成正比;Ｏ３暴露２ｈ使ＭＤＡ含量和ＧＳＳＧ含量明显增加,ＧＳＨ减少;ＶＩＰ预处理呈剂量依赖性降低Ｏ３暴露的ＣＩ值、降低ＭＤＡ和ＧＳＳＧ含量、增加ＧＳＨ及ＧＳＨ／ＧＳＳＧ比值、增加ＣＡＴ活性,显示出细胞保护效应;ＶＩＰ的保护效应可被放线菌素Ｄ（Ａ－Ｄ）或蛋白激酶Ｃ阻断剂Ｈ７部分取消。结果表明：Ｏ３暴露会导致ＢＥＣ损伤,ＶＩＰ可通过增强ＢＥＣ的抗氧化能力而保护ＢＥＣ,ＶＩＰ的信号在细胞内的转导途径与基因转录及依赖ＰＫＣ的酶蛋白磷酸化有关。     

小牛类表皮生长因子活性肽（c－EGF）的分离纯化及性质

本文报道小牛颌下腺通过酸抽提、ＣＭ５２、ＤＥ５２柱层析、ＳｅｐｈａｄｅｘＧ７５凝胶过滤及ＨＰＬＣ等步,分离纯化到一个分子量为６ｋＤ,等电点为４．６的小肽。该小肽类似人重组表皮生长因子（ｒｈ—ＥＧＦ）的生物活性,具有刺激ＮＲＫ细胞的增殖及刺激新生小鼠的睁眼、萌牙,并具有增加Ａ４３１细胞膜蛋白质磷酸化的功能。氨基酸组成缺少苯丙氨酸及苏氨酸残基。我们命名它为小牛类ＥＧＦ样活性肽（ｃａｌｆＥＧＦ－ｌｉｋｅａｃｔｉｖｅｐｅｐｔｉｄｅ,简称ｃ－ＥＧＦ）。     

血管活性肠肽对肺表面活性物质结合蛋白A表达的影响

目的:研究血管活性肠肽(VIP)对肺表面活性物质结合蛋白A(SP-A)表达的影响以及VIP调控SP-A表达的细胞内信号转导途径.方法:运用免疫组织化学和RT-PCR技术研究VIP对SP-A表达的影响;并进一步运用受体拮抗、蛋白激酶抑制、反义寡核苷酸阻断等手段探讨VIP促进SP-A表达的信号转导途径.结果:①VIP(10-8mol/L)促进肺泡Ⅱ型细胞(ATⅡ)细胞中的SP-A蛋白表达和提高肺组织SP-AmRNA含量:②VIP受体拮抗剂(10-6mol/L)可取消VIP(10-8mol/L)促进SP-A表达的效应;③蛋白激酶C抑制剂H7(10-5mol/L)和c-fos基因的反义寡核苷酸(9×10 6mol/L)均可阻断VIP促进SP-A表达的作用.结论:VIP通过其受体促进SP-A的表达,PKC及c-fos蛋白在介导VIP促进SP-A表达的细胞内信号转导过程中起重要作用.     

发育过程中肝脏血管活性肠肽及其受体量的变化

已有的研究观察到,胚胎肝脏中血管活性肠肽(vasoactiveintestinalpolypeptide,VIP)及其受体(vasoactiveintesti-nalpolypeptidereceptor,VIPR)与造血干细胞生长和肝脏发育有关。本研究旨在了解发育过程中肝VIP及VIPR量的动态变化。采用放射免疫分析法、生物分子相互作用系统和RT-PCR等技术检测了各发育阶段大鼠肝组织VIP浓度、VIP受体结合量及VIP受体表达亚型,实验观察到胎鼠和新生鼠肝脏VIP浓度显著低于未成年鼠及成年鼠肝脏VIP浓度(P<0.05)。发育尚未成熟时(胎鼠、新生鼠、未成年鼠),肝VIPR表达均明显高于成年鼠(P<0.05),表明大鼠在发育过程中肝脏VIP与VIP受体量呈相反的变化趋势。大鼠发育各时期,肝脏均表达VIPR-1。这些结果部分解释了肝脏发育、肝脏造血转移等重要生理现象。     

高血压病患者血浆胰岛素与血管活性肠肽水平的改变

高血压病患者血浆胰岛素与血管活性肠肽水平的改变徐珞苏海灵王瑞华１唐明（青岛医学院生理学教研室,青岛２６６０２１;电力工业部青岛培养院）高血压是心血管病的主要危险因素。近年来研究表明,胰岛素（Ｉｎｓ）可能是高血压发病的重要机制之一。为了探讨Ｉｎｓ与血管...     

血管活性肠肽的免疫调节作用

血管活性肠肽作为神经和内分泌系统中一种多功能的神经递质和神经调节因子,在上述两个生理系统中发挥重要的调节作用;同时也对机体免疫系统起着重要的作用,尤其是在局部黏膜免疫中起着一定的调节作用。血管活性肠肽通过它的两个受体VPAC1和VPAC2发挥生物效应。     

猕猴发育过程中肠肝组织血管活性肠肽及其受体的变化

本文旨在探讨猕猴发育过程中血管活性肠肽(vasoactive intestinal polypeptide,VIP)及其受体在肠肝组织的变化。通过手术途径获得胚胎6月、新生2 d、新生45 d和成年猕猴的回肠、肝脏、门静脉和外周血等标本,应用放射免疫分析法测定各标本中的VIP含量;通过免疫组化方法观察VIP在肠、肝组织内的分布;利用原位杂交法检测VIP受体1(VIP receptor 1,VIPR1)的表达。结果显示:(1)胚胎6月的猕猴小肠VIP含量为(20．7±14．3)ng／mg蛋白;小肠绒毛根部及黏膜下层可见少量的VIP阳性染色颗粒;在发育过程中,小肠VIP含量逐渐增加,成年期时达(514．8±49．2)ng／mg蛋白,较胚胎6月显著增加(P<0．01)。(2)成年猕猴小肠VIP主要分布于绒毛隐窝部、黏膜下层神经及环、纵行肌间神经丛及环行肌,在发育过程中相应部位的VIPR1表达逐渐上调。(3)肝脏在发育过程中VIP及VIPR1含量逐渐降低。(4)发育的各个时期,小肠组织的VIP含量均明显高于肝脏组织,门静脉VIP水平也始终高于外周血。结果提示,小肠绒毛隐窝部、黏膜下层神经及环、纵行肌间神经内VIP及VIPR1含量足在出生以后才迅速增加的;不论是在胚胎还是成年期,VIP均不在肝中代谢和分解,VIPR1仅见于胚胎肝脏血管。     

Vasoactive intestinal peptide enhances wound healing and proliferation of human bronchial epithelial cells

In the present study, we investigated the effects of vasoactive intestinal peptide (VIP) on wound healing of bronchial epithelium. Wound healing of the mechanical damaged human bronchial epithelial cells (HBEC) was observed in the absence or presence of VIP. Effects of VIP on chemotactic migration, cell proliferation of HBEC were also tested. HBEC chemotaxis was assessed by the blind well chamber technique, the cell cycle was determined by flow cytometry, and cell proliferation was determined by measuring the expression of proliferating cell nuclear antigen Ki67. Effects of VIP on epithelial E-cadherins protein and mRNA were also measured by immunohistochemistry and RT-PCR. The results showed that VIP accelerated the recovery of wound area of HBEC. VIP increased the migration and proliferation of HBEC, and these effects were blocked by a VPAC1 receptor antagonist. VIP also increased the expression of E-cadherin mRNA and protein in HBEC, suggesting that protective effects of VIP on wound healing may be related to its ability to increase the expression of E-cadherin. In conclusion, VIP has protective effects against human bronchial epithelial cell damage, and the beneficial effects of VIP might be mediated, at least in part, by VPAC1, and associated with increased expression of E-cadherin.     

纤维连接蛋白上调兔支气管上皮细胞过氧化氢酶表达

为从基因转录水平阐明纤维连接蛋白(fibronectin,Fn)与整合素(integrins)结合反应对支气管上皮细胞(bronchial epithelialCells,BECs)的抗氧化保护机制,本文在先前的工作基础上用臭氧(ozone,O_3)攻击原代培养的免BEC,RT-PCR扩增过氧化氢酶(catalase,CAT)的cDNA,PCR产物经琼脂糖凝胶电泳后用凝胶成像系统进行灰度分析,反映CAT mRNA的原始表达丰度,观察Fn处理的影响及蛋白酪氨酸激酶抑制剂genistein和钙调素抑制剂W_7的作用。同时,将电泳展开的PCR产物电转移至尼龙膜上,用CAT特异性寡核苷酸探针杂交,证实PCR扩增产物为特异性目的基因的转录产物。结果证实:Fn(10μg/ml)处理可提高CAT表达(P<0.01),蛋白酪氨酸激酶抑制剂genistein可阻断Fn对CAT mRNA表达的增强效应(P<0.01);钙调素抑制剂W_7对Fn处理后CAT mRNA表达增强也有抑制作用。提示:Fn可提高BEC细胞内CAT编码基因的转录水平,其上游信号途径与整合素介导的酪氨酸磷酸化或Ca~(2+)-钙调素通路有关。     

Vasoactive intestinal peptide: levels and functional receptors in rat brain before and after weaning.

The average level of VIP was found to be 17 pmol/g wet weight in the brain of the newborn rat. This level ramained constant during the first two weeks after birth then increased progressively to 40 pmol/g wet weight at 20 days, a value comparable to that found in adult animals. VIP was already able to stimulate brain membrane adenylate cyclase activity at birth. The stimulation with 10^?6 M VIP allowed a 2.5-fold increase in basal activity in membranes from 1 to 14-days-old pups as compared to a 1.7-fold stimulation in membranes from adult brain. The apparent activation constant for VIP adenylate cyclase stimulation was 4.10^?7 M at all ages. The efficiency of VIP activation amounted to as much as 70% of that of fluoride at birth and to 35% only of fluoride activation in brain membranes from adult rats.     

Epidermal growth factor stimulates collagen synthesis in liver-derived epithelial clone cells

The effect of epidermal growth factor (EGF) on collagen fiber formation in clone RLC-18(4) epithelial cells obtained from rat liver was investigated by silver impregnation and assay of hydroxyproline content. EGF caused dose-related stimulation of collagen fiber formation and was effective at as low as concentration as 0.5 ng/ml. Actinomycin D suppressed collagen fiber formation increased by EGF, suggesting that this factor stimulates de novo collagen synthesis in the cells.     

Activation of epidermal growth factor receptor via CCR3 in bronchial epithelial cells

We have previously found that bronchial epithelial cells express CCR3 whose signaling elicits mitogen-activated protein (MAP) kinase activation and cytokine production. Several investigators have focused on the signaling crosstalk between G protein-coupled receptors (GPCRs) and epidermal growth factor receptor (EGFR) in cancer cells. In this study, we investigated the role of EGFR in CCR3 signaling in the bronchial epithelial cell line NCI-H292. Eotaxin (1-100 nM) induced dose-dependent tyrosine phosphorylation of EGFR in NCI-H292 cells. Pretreatment of the cells with the EGFR inhibitor (AG1478) significantly inhibited the MAP kinase phosphorylation induced by eotaxin. Eotaxin stimulated IL-8 production, which was inhibited by AG1478. The transactivation of EGFR through CCR3 is a critical pathway that elicits MAP kinase activation and cytokine production in bronchial epithelial cells. The delineation of the signaling pathway of chemokines will help to develop a new therapeutic strategy to allergic diseases including bronchial asthma.     

Liver epithelial cell migration induced by epidermal growth factor or transforming growth factor alpha is associated with changes in the gene expression of secreted proteins

Summary Rat liver epithelial cells are induced to migrate by epidermal growth factor (EGF) or transforming growth factor alpha (TGF-α) in serum-free medium supplemented with insulin. Immunohistological staining of the migration tracks containing laminin and fibronectin has allowed a quantitative analysis of the process. The growth factor-induced migration is relatively slow, but very efficient. Between 24 and 48 h after exposure to EGF (or TGF-α), 50 to 70% of the cells have migrated away from their site of initial attachment and spreading. This delayed effect of the interaction of the receptor with its ligands is associated with changes in gene expression, but is not associated with a stimulation of cell proliferation. In serum-free medium supplemented with insulin, the cells secrete six major proteins, as revealed by SDS-polyacrylamide gel electrophoresis. The media of cultures supplemented with insulin plus EGF (or TGF-α) contain in addition two new proteins and an increased amount of fibronectin. One secreted protein is synthesized in significantly reduced amounts. The most conspicuously EGF-induced protein (EIP-1, Mr 47 000) is detected within 2 h, depends on the continued presence of the growth factor, and has not been detected as bound to the substratum. The stringent regulation of EIP-1 suggests that this gene product might participate in the modulation of the changes induced by the growth factor. The system is being used for the further analysis of the regulation of gene expression by EGF and of the migration of normal and neoplastically transformed epithelial cells. This paper is dedicated to the memory of Dr. Luis F. Leloir. A preliminary communication has been presented at the Cold Spring Harbor Meeting on Liver Gene Expression, May 1987. Editor's Statement Mitogen-stimulated gnees are an active area of study with fibroblastic systems. In this paper the approach is extended to epithelial cells and functional correlations are also made.     

Vasoactive intestinal peptide induces IL-8 production in human colonic epithelial cells via MAP kinase-dependent and PKA-independent pathways

Vasoactive intestinal peptide (VIP) has been shown to be a key regulator of intestinal epithelial functions such as mucus and chloride secretion, paracellular permeability, and cell proliferation. However, its regulatory role in intestinal epithelial chemokine production remains unknown. The aim of this study was (1) to determine whether VIP can modulate intestinal epithelial interleukin-8 (IL-8) production and (2) to identify intracellular mediators responsible for this effect. In the human colonic epithelial cell line HT29-Cl.16E, VIP stimulates IL-8 secretion dose-dependently and IL-8 mRNA level at 10(-9) M. The protein kinase A (PKA) inhibitor PKI did not abolish the effect of VIP. However, inhibition of the ERK1/2 and p38 MAPK pathways reduced the VIP-stimulated IL-8 secretion and mRNA level. Together, our results showed that VIP stimulates IL-8 production in intestinal epithelial cells via PKA-independent and MAPK-dependent pathways. These data suggest that VIPergic pathways can play an immunomodulatory role in intestinal epithelial cells, by regulating epithelial IL-8 secretion.     

Bronchioalveolar morphogenesis of human bronchial epithelial cells depending upon hepatocyte growth factor

Lung alveolar regeneration occurs in adult human lungs as a result of proliferation, differentiation and alveolar morphogenesis of stem cells. It is increasingly being believed that bronchial epithelial cells (BECs) have a potential as stem cells, because they are potent to differentiate into multiple central and peripheral lung cell types in three‐dimensional (3D) cultures, and they develop multiple foci with well‐differentiated histogenesis after transformed into neoplastic cells. In this study, we investigated morphogenic abilities of HBE135 human BECs immortalized by E6/E7 oncogene in 3D cultures. When HBE135 cells were cultured alone or co‐cultured with endothelial cells, the cells formed spherical colonies without branching. However, in co‐culture with lung fibroblast MRC‐9 cells, HBE135 cells formed colonies with bronchioalveolar‐like complex branching, suggesting that MRC‐9‐derived soluble factor(s) are responsible for the branching formation. MRC‐9 cells, not endothelial cells, were found to highly express hepatocyte growth factor (HGF), a soluble molecule involved in liver and kidney regeneration. An anti‐HGF neutralizing antibody severely suppressed the complex branching formation, but addition of HGF could not sufficiently compensate the morphogenic effects of MRC‐9 cells, suggesting that MCR‐9‐derived HGF was necessary but insufficient for the bronchioalveolar structure formation. Immunohistochemistry revealed that Met, a cognate receptor for HGF, was highly expressed and phosphorylated in neoplastic BECs from lung adenocarcinomas with well‐differentiated, not poorly differentiated, histogenesis. These results are consistent with the notion that BECs have an aspect of stem cells. This aspect appears to become manifest through HGF–Met signalling pathway activation.