人表皮细胞生长因子及其研究进展

表皮细胞生长因子是人体内一种重要自分泌/旁分泌的生长因子。人表皮细胞生长因子及其受体调控细胞增殖、分化、迁移、生存等与细胞命运密切相关的过程,在细胞发育、伤口愈合、器官发生中发挥重要作用。本文将对人表皮细胞生长因子的合成、分泌、生化特性、分子结构、家族、受体、信号通路、生物学活性、生理功能、应用及制备方法等方面进行综述。     

表皮生长因子受体基因在中国人部份肿瘤细胞株中的扩增

<正> 肿瘤基因扩增是肿瘤基因激活的一种方式。而表皮生长因子受体基因在结构上作为v-erb-B的同源基因;在功能上其产物与src基因家族产物有相同的作用。因此,它的扩增与否与肿瘤的发生发展密切相关。因为肿瘤基因的激活,能导致肿瘤基因产物的异常表达。而表皮生长因子受体基因及表达产物的异常,又可能是细胞恶性增殖、低度分化的重要原因之一。 为了比较中国人常见的肿瘤细胞株表皮生长因子受体基因在DNA水平上的扩增状态,本研究选用了九种不同的肿瘤细胞株,用分子杂交的方法进行了研究。     

表皮生长因子受体

表皮生长因子受体（EGFR）是一种具有酪氨酸激酶活性的膜表面受体,其胞内区的3个亚区是其发挥酪氨酸激酶活性、介导信号转导的关键部位。表皮生长因子受体和其他的erbB受体可形成同源和异源的多种二聚体,不同的二聚体与表皮生长因子受体的6种配体形成的不同组合可将不同的细胞外刺激传入胞内。表皮生长因子可激活多种下游信号路径,产生多种生物学效应,ras-raf-MEK-erk/MAPK途径与增殖的激活有关,PI3K－PKC－IKK途径与细胞移动性的增强有关。表皮生长因子受体与肿瘤的发生发展和器官的修复有密切的关系,针对表皮生长因子受体的肿瘤治疗和器官修复具有良好的应用前景。     

腺苷A2A受体在小鼠颅脑创伤与外周组织损伤模型中的作用差异

本文旨在探索腺苷A2A受体在颅脑创伤、皮肤创伤及放射损伤复合创伤中的作用差异.分别观察和检测野生型小鼠、A2A受体基因敲除小鼠以及给予A2A受体激动剂CGS21680治疗的小鼠在皮肤创伤、放射损伤复合创伤后的伤口愈合时间以及颅脑创伤后的神经功能缺损情况、伤侧皮层脑含水量、脑脊液中谷氨酸浓度.结果表明,CGS21680促进外周组织伤口愈合,却加重颅脑创伤模型的神经功能损害,这与其促进谷氨酸释放有关.相反,A2A受体基因敲除显著延迟小鼠皮肤创伤及放射损伤复合创伤模型的伤口愈合,而在颅脑创伤模型中通过抑制谷氨酸释放产生保护效应.本研究初步证实,A2A受体激活促进谷氨酸大量释放可能是其在中枢损伤与外周损伤产生作用差异的机理之一,这为将来临床应用A2A受体激动剂减轻外周损伤,而用A2A受体拈抗剂减轻颅脑损伤提供了一定的实验依据.     

生长因子在骨折愈合过程中作用的研究进展

骨折愈合是一个复杂的病理生理过程,涉及多种细胞和生长因子的协同作用。随着分子生物学和基因工程的发展,生长因子在骨折愈合过程中的基础研究和临床应用取得了较大的进展。本文就转化生长因子β．碱性成纤维细胞生长因子、血小板衍生生长因子、表皮生长因子、神经生长因子等五种生长因子在骨折愈合过程中的作用及近年来的研究进展作一综述,并分析五种生长因于在法医学中的应用前景。     

表皮生长因子受体在正常大鼠眼组织中的表达

采用免疫组织化学方法观察了表皮生长因子受体在正常大鼠眼组织的表达。结果发现：角膜上皮的深层细胞和角膜缘上皮细胞、部分结膜上皮细胞和结膜下结缔组织内成纤维细胞均强烈表达表皮生长因子受体。结果显示：表皮生长因子受体阳性细胞主要分布于眼表层组织。这些细胞不仅是眼组织损伤后修复、而且是多种手术能否成功和某些疾病形成中起重要作用的细胞     

适量的生长因子能治愈创伤和骨伤

Louisville大学生物化学系副教授Gregory Schultz说,生长因子能加速人伤口和骨骼的愈合。这一证明是许多大型制药公司一直期待、而现在仍面临的问题。去年年底,在技术管理小组(TMG)发起的“生长因子对伤口愈合”的评论会上,schultz认为,供给系统对于商品化这些大型、不稳定(由细胞分泌并能诱导细胞增殖)的蛋白质是非常重要的。目前,大多数研究人员采用常规基质例如凝胶和乳脂,将生长因子用于创伤表皮。而其     

Notch与相关调控信号的相互作用在伤口愈合中的调控

皮肤是哺乳动物最重要的组织之一。当皮肤受损时,受损组织通过系列伤口愈合反应的生理和心理作用被修复,实现组织再生。再生反应主要发生在胚胎发育早期,伤口自愈能力随着机体的成熟而减弱;并且哺乳动物的组织重塑过程较为复杂,在不正确的信号引导下,可能引起并发症而导致创面愈合异常。研究表明,伤口微环境的稳态和信号分子的辅助作用是愈合的重要因素。Notch作为重要的信号通路,参与调控上皮巨噬细胞募集和血管内皮细胞再生等伤口愈合阶段。Wnt信号促进伤口表皮干细胞的增殖和血管网络结构的形成。Hedgehog信号驱动伤口处毛囊发育及其周围组织再生,TGF-β信号有助于愈合时多细胞层形成和瘢痕减少。本文重点阐述Notch信号通路以及Notch与相关信号分子互作在伤口愈合中的调控作用,旨在总结信号分子在组织工程研究方面的最新进展,分析伤口微环境的信号互作机制,为长时间伤口愈合和过度伤口愈合的治疗提供理论依据。     

大脑中的又一新发现

苏联杂志《知识就是力量》报道,苏联医学科学院全苏心脏病治疗中心的学者们在哺乳动物(包括人)的大脑里发现了一种专门“负责”愈合伤口的生理系统。他们指出,感受体的疼痛信号传输给大脑特别组织后,大脑组织很快就在有机体内产生一种能止痛和加快愈合伤口的物质。据验证,是大脑里的一种叫“激活”的物质对促使皮肤伤口愈合所起的作用。该治疗中心的学者在白鼠身上做试验,他们把电极埋藏于白鼠的灰体中心部位,一周后,在每只白鼠的背部作     

消溃灵对大鼠胃溃疡愈合时粘膜EGFR表达的影响

为了研究中药消溃灵对消化性溃疡愈合的影响及其对胃粘膜保护作用,采用乙酸注射至大鼠胃窦部浆膜下形成慢性胃溃疡模型,测定溃疡指数,并用免疫组织化学的方法,观察表皮生长因子受体在溃疡周围粘膜的表达并进行图像分析。结果表明雷尼替丁组和消溃灵组溃疡指数显著低于模型组（Ｐ＜０．０１）。模型组和雷尼替丁组表皮生长因子受体较对照组增加（积分光密度值和阳性细胞占总面积的百分比为Ｐ＜０．０１）,消溃灵组比模型组和雷尼替丁组进一步增加（积分光密度值为Ｐ＜０．０５,阳性细胞占总面积的百分比为Ｐ＜０．０１）。本文提示,消溃灵对表皮生长因子受体的表达有一定的促进作用,这可能是消溃灵促进胃溃疡愈合和胃粘膜保护作用的机理之一。     

Accumulation of human EGF in nectar of transformed plants of Nicotiana langsdorffii x N. sanderae and transfer to honey by bees

Honey has been used successfully in wound healing for thousands of years. The peptide hormone human epidermal growth factor (hEGF) is also known to have a beneficial effect in various wound healing processes via mechanisms that differ from those for honey. In this study, we show that hEGF can be incorporated into honey via nectar. Plants of Nicotiana langsdorffii × N. sanderae were transformed with the gene for hEGF, equipped with a nectary‐targeted promoter and a signal sequence for secretion to nectar. These plants accumulated hEGF in the nectar. The maximum hEGF concentration recorded with ELISA in these plants is 2.5 ng·ml^?1. There is a significant linear relationship (P < 0.001) between hEGF concentration and induction of hEGF‐receptor phosphorylation. Since the flower morphology of these plants did not allow production of honey from their nectar, we used feeding solutions, spiked with synthetic hEGF, to study transfer of this peptide into honey through bee activity. Transfer of hEGF from a feeding solution to honey by bees occurred with retention of the hEGF concentration and the capacity to induce hEGF‐receptor phosphorylation. These observations indicate that plants can function as a production platform for honey containing biologically active peptides, which may enhance wound healing and other biological processes.     

Expression of a fusion protein containing human epidermal growth factor and the collagen-binding domain of <Emphasis Type="Italic">Vibrio mimicus</Emphasis> metalloprotease

Human epidermal growth factor (hEGF) is a polypeptide of 53 amino acids, is an important autocrine/paracrine factor in the human body, and is used in the pharmaceutical and cosmetics industries. We constructed a fusion hEGF protein with a collagen-binding domain (CBD) composed of 33 amino acids from Vibrio mimicus metalloprotease (VMCBD). The CBD segment of the metalloprotease was fused at the C terminus of the hEGF protein. The recombinant fusion protein was expressed in Escherichia coli and purified. The purified hEGF protein promoted greater growth of human/A-431 cells than did the control hEGF. The fusion EGF protein also showed collagen-binding activity with type I collagen. In contrast, hEGF did not bind to type I collagen. These results suggest that recombinant hEGF protein fused to VMCBD may be able to remain for a long period at injured epidermal tissue acting as a healing agent.     

Synthesis and secretion of an epidermal growth factor (EGF) by human fibroblast cells in culture

Human fibroblast (WS-1) cells in culture synthesized and secreted an epidermal growth factor which cross-reacted with human epidermal growth factor (hEGF) purified from human urine. hEGF secreted by the cells (designated as WS-1 EGF or fibroblast EGF) and hEGF isolated from urine (designated as urine EGF) were immunologically indistinguishable. The molecular weight of fibroblast EGF estimated by gel filtration was identical with that of hEGF from urine. On chromatofocusing chromatography, fibroblast EGF was eluted mainly at pH 4.26 as a sharp symmetric peak with a minor peak at pH 4.62, like urine EGF. These results suggested that EGF synthesized and secreted by human fibroblast cells is an identical molecule to that of hEGF in human urine.     

Receptor-Purified,Bolton-Hunter Radioiodinated,Recombinant, Human Epidermal Growth Factor: An Improved Radioligand for Receptor Studies

Abstract

We report an assessment of the applicability of the Bolton-Hunter method to the radioiodination of epidermal growth factor (EGF). Recombinant human EGF (hEGF) could be radioiodinated successfully by this method, whereas murine EGF could not. Bolton-Hunter ¹²⁵I-labeled hEGF was compared with commercial ¹²⁵I-labeled hEGF prepared by the chloramine-T radioiodination method. Neither radioligand was sufficiently pure for a detailed characterization of the purportedly heterogeneous pattern of binding of EGF to its receptors. A procedure based on receptor adsorption was thus developed for repurification of the Bolton-Hunter ¹²⁵I-labeled hEGF. This provided a much purer radioligand suitable for detailed studies of receptor-binding heterogeneity.     

Complexes of telomeric oligonucleotides with the PGEk protein vector: Internalization by target cells and antiproliferative activity

Recombinant protein PGEk, consisting of the human epidermal growth factor (hEGF) and a DNA-binding domain, proved to be capable of interacting with the hEGF receptor and inducing cell proliferation, as characteristic of hEGF. PGEk complexes with the telomere-mimic oligonucleotide d(TTAGGG)₄ (TMO) and its thio analog (TMS) were efficiently and selectively internalized by cells with a high-level expression of the hEGF receptors. The extent of internalization was studied as a function of the PGEk: oligonucleotide ratio in the complex. The intracellular location of the oligonucleotides was determined. PGEk was found to ensure a more efficient delivery of the oligonucleotides and to protect them from nuclease degradation. The oligonucleotides contained in the complexes exerted a far greater cytotoxic effect as compared with the free oligonucleotides.     

Partial purification properties of human epidermal growth factor from recombinant Escherichia coli by expanded bed adsorption

Human epidermal growth factor is a polypeptide hormone having many diverse biological functions. This paper first presents the recovery results of human epidermal growth factor (hEGF) immediately from the fermentation broth of recombinant Escherichia coli by using an expanded bed system (a couple of STREAMLINE25 and ÄKTA explorer 100). The influences of operational conditions such as linear flow rate, gradient length of NaCl concentration, pH and sample concentration on the purification performances of hEGF in expanded and packed bed modes with STREAMLINE DEAE resin were systematically evaluated. After optimization, the practical recovery procedure in the expanded bed mode was carried out on a scaled-up system under the conditions of linear flow rates of 183 cm/h (upward) and 37 cm/h (downward), sample volume of 300 ml and column bed height of 13.8 cm which yielded a primary product of hEGF from the cell-free supernatant containing hEGF after centrifugation at 4000 rev/min for 15 min. As a result, the hEGF concentration in the product was higher than 20% (w/v), the concentration factor was greater than 4.3 and the total yield was higher than 80%, respectively. At the same time, the results of hEGF recovery by using expanded bed adsorption (EBA), packed bed chromatography (PBC) and salting out were compared. The results show that the procedure of hEGF recovery in expanded bed adsorption has some advantages over the other two procedures, because of its higher concentration factor, recovery yield, productivity, hEGF concentration in the primary product and shorter duration of purification run.     

Production and properties of a monoclonal antibody against human epidermal growth factor

A monoclonal antibody against human epidermal growth factor (hEGF) was obtained from a mouse hybridoma cell line. The purified monoclonal antibody from the ascites fluid of a mouse injected with one of the cell lines was specific for hEGF and did not cross-react with mouse EGF (mEGF). Its Kd value for hEGF was 1.4 X 10(-9) M. This monoclonal antibody inhibited the biological activities of hEGF, including its binding to the receptor of BALB/3T3 cells and its stimulation of DNA synthesis in the cells, but did not affect the activities of mEGF. The monoclonal antibody completely inhibited DNA synthesis stimulated by human urine from a patient without a tumor, but only partially inhibited the stimulatory activity in urine from a tumor-bearing patient.     

Characterization of a high-molecular-weight form of epidermal growth factor in an extract of human urine

We purified from a side fraction of the commercial preparation of urokinase from large volumes of human urine a high-molecular-weight (HMW) form of human epidermal growth factor (hEGF). Sequence analysis of the amino terminus of the intact molecule and of two tryptic fragments and carboxypeptidase Y analysis revealed the molecule to correspond to residues 828-1023 of the hEGF precursor predicted by the nucleotide sequence of human renal hEGF mRNA, with hEGF forming its carboxyl terminus. HMW hEGF bound poorly to concanavalin A-agarose, quite avidly to wheat germ lectin-agarose, and completely to phenyl boronate-agarose, suggesting that it was O-glycosylated. Sephacryl S-200 chromatography of freshly-voided urine revealed mostly hEGF, with smaller amounts of a much higher molecular weight hEGF, but little material that was the size of the HMW hEGF we characterized. The large fragment we characterized presumably is cleaved from the larger form by enzyme(s) present in urine during the collection, storage, and processing of urine. We have confirmed that hEGF is synthesized as a large precursor molecule, as predicted by the nucleotide sequence of hEGF mRNA.     

EGF and TGF-alpha in wound healing and repair

Wound healing is a localized process which involves inflammation, wound cell migration and mitosis, neovascularization, and regeneration of the extracellular matrix. Recent data suggest the actions of wound cells may be regulated by local production of peptide growth factors which influence wound cells through autocrine and paracrine mechanisms. Two peptide growth factors which may play important roles in normal wound healing in tissues such as skin, cornea, and gastrointestinal tract are the structurally related peptides epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). EGF/TGF-alpha receptors are expressed by many types of cells including skin keratinocytes, fibroblasts, vascular endothelial cells, and epithelial cells of the GI tract. In addition, EGF or TGF-alpha are synthesized by several cells involved in wound healing including platelets, keratinocytes, and activated macrophages. Healing of a variety of wounds in animals and patients was enhanced by treatment with EGF or TGF-alpha. Epidermal regeneration of partial thickness burns on pigs or dermatome wounds on patients was accelerated with topical application of EGF or TGF-alpha, and EGF treatment accelerated healing of gastroduodenal ulcers. EGF also increased tensile strength of skin incisions in rats and corneal incisions in rabbits, cats, and primates. Additional research is needed to better define the roles of EGF, TGF-alpha and their receptor in normal wound healing, to determine if alterations have occurred in the EGF/TGF-alpha system in chronic wounds, and optimize vehicles for effective delivery of peptide growth factors to wounds.