肝素结合表皮生长因子及其在恶性肿瘤中的作用

肝素结合表皮生长因子属表皮生长因子超家族成员之一,可促进多种细胞分裂和增殖,参与多个病理生理过程。近年研究发现肝素结合表皮生长因子可参与肿瘤发生发展的各个环节,在肿瘤化疗耐药中也发挥一定作用,使其成为恶性肿瘤生物学治疗的靶向因子。其与白喉毒素结合的特性为临床提供了一种新的治疗癌症的途径。继续对HB-EGF进行深入的研究,并将已获得的知识与临床治疗结合,将有望产生新的治疗恶性肿瘤的途径及新型的抗肿瘤药物。     

系统表达HB-EGF转基因小鼠的建立

目的建立系统表达肝素结合性表皮生长因子（HB-EGF）的转基因动物模型,利用转基因动物模型研究HB-EGF与组织纤维化的关系。方法RT-PCR法克隆小鼠HB-EGF基因,将其插入Chickenβ-actin启动子下游,构建Chickenβ-actin-HB-EGF表达载体,利用显微注射的技术把表达载体注射到受精卵的雄原核中,建立HB-EGF转基因小鼠。利用特异引物PCR的方法鉴定转基因的基因型,采用Western Blot方法鉴定HB-EGF基因在全身组织的表达。分别取HB-EGF转基因鼠与同窝阴性小鼠的肝、肾、肺及膀胱组织进行Massion染色。结果建立了系统表达HB-EGF转基因小鼠,Western Blot发现其HB-EGF在肝、肺、肾及膀胱的表达与同窝阴性对照小鼠相比明显增加。Massion染色结果表明转基因鼠肝、肺、肾及膀胱组织纤维化程度明显高于同窝阴性对照小鼠。结论成功建立了系统表达HB-EGF转基因小鼠,HB-EGF的过度表可显著加重组织纤维化程度。     

人参皂甙Rb1抑制扩张型心肌病发生中的HB-EGF表达和纤维化

目的HB-EGF过表达可促进心肌纤维化及心肌细胞凋亡,本文研究人参皂甙Rb1对cTnT^R141W转基因扩张型心肌病小鼠发病过程中的HB-EGF表达和心肌纤维化的影响。方法将cTnT^R141W转基因小鼠随机分为模型组和人参皂甙Rb1组（70 mg/kg/d）,连续给药7个月,取野生型小鼠作为对照组。用Kaplan-Meier法进行生存分析。心脏超声检测心功能及心脏几何构型。计算心重指数。光镜观察心肌细胞及间质变化。Western blot检测心脏HB-EGF,pSTAT3表达水平。结果Rb1长期给药能显著改善该模型的心功能和心脏几何构型,将死亡率降低50%。Rb1治疗组心重指数降低11.3%（P〈0.05）,光镜观察显示Rb1能减轻心肌细胞排列紊乱以及间质纤维化。Western blot结果显示Rb1能够显著降低模型中的HB-EGF及pSTAT3的表达。结论Rb1抑制心肌病发生中的HB-EGF表达及抑制下游信号pSTAT的激活,并改善扩张型心肌病模型的心功能及心脏重构。     

肝素结合表皮生长因子抑制剂CRM197 对裸鼠卵巢癌移植瘤 的抑制作用

目的:研究CRM197对裸鼠卵巢癌移植瘤生长的抑制作用探讨其对逆转卵巢癌组织耐药作用及其在卵巢癌治疗中的可行性.方法:用人卵巢癌亲本细胞A2780、耐紫杉醇细胞A2780/Taxol及耐顺铂细胞A2780/DDP分别建立裸鼠卵巢癌移植瘤模型,观察注射CRM197组与阴性对照组裸鼠肿瘤生长情况,免疫组化法测定各组裸鼠肿瘤组织中HB-EGF及P-gp表达情况.结果:HB-EGF及P-gp在各肿瘤组织中不同程度的表达,在注射CRM197的裸鼠肿瘤组织中表达明显降低(P＜0.05),差异具有统计学意义.结论:CRM197通过抑制肝素结合表皮生长因子的信号传导通路激活抑制了裸鼠卵巢癌移植瘤的生长,使裸鼠卵巢癌移植瘤HB-EGF及P-gp表达明显降低.     

EGF类生长因子来源的新型靶向肽在抗肿瘤药物蛋白中的应用

许多肿瘤细胞表面表皮生长因子受体EGFR都存在过表达现象。考察了牛痘病毒生长因子(VGF)中的EGFR结合域(S3)与人的肝素样表皮生长因子(HB-EGF)来源的肝素结合域(命名为HE)重组后对肿瘤细胞的选择性。通过重组表达带有靶向和穿膜结构域的EGFP-S3-HE和EGFP-S3-HE-TATm两种融合蛋白与正常细胞和肿瘤细胞的共孵育实验来研究其对肿瘤细胞的特异性靶向吸附和穿膜效应。进一步将S3-HE-TATm靶向穿膜序列与苦瓜来源的核糖体失活蛋白MAP30融合,可显著提高MAP30对肿瘤细胞的抑杀作用,但这种抑杀作用却对正常细胞仍保持在较低水平。由此表明S3-HE-TATm是一种新型优异的肿瘤细胞靶向药物运输载体,可用于肿瘤治疗的进一步开发研究。     

卵巢癌患者血清HB-EGF、TK1、GDF15水平与临床病理特征和预后的关系

摘要 目的：研究卵巢癌患者血清肝素结合性表皮生长因子（HB-EGF）、胸苷激酶1(TK1)、生长分化因子15(GDF15)水平与临床病理特征和预后的关系。方法：利用酶联免疫吸附试验（ELISA）检测94例卵巢癌患者和60例健康体检志愿者的血清HB-EGF、TK1、GDF15水平。Pearson相关分析卵巢癌患者血清HB-EGF、TK1、GDF15三者的相关性。分析卵巢癌患者血清HB-EGF、TK1、GDF15水平与临床病理特征的关系。Kaplan-Meier生存分析不同血清HB-EGF、TK1、GDF15水平的卵巢癌患者的生存率差异。单因素及多因素COX回归分析影响卵巢癌患者生存预后的因素。结果：与健康对照组相比,卵巢癌组患者血清HB-EGF、TK1、GDF15水平明显较高（均P<0.05）。卵巢癌组患者血清HB-EGF与TK1、GDF15水平呈正相关,TK1与GDF15水平呈正相关（均P<0.05）。卵巢癌患者血清HB-EGF、TK1、GDF15水平与FIGO分期、分化程度有关（均P<0.05）。血清HB-EGF、TK1、GDF15高水平的卵巢癌患者3年总体生存率分别低于低水平患者（P<0.05）。血清HB-EGF、TK1、GDF15高水平、FIGO分期为Ⅲ期及低分化程度是影响卵巢癌患者预后的独立危险因素。结论：卵巢癌患者血清中HB-EGF、TK1、GDF15水平升高,三者水平与卵巢癌肿瘤FIGO分期、肿瘤分化程度有关,检测血清HB-EGF、TK1、GDF15水平有助于评估卵巢癌患者的预后。     

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。     

心肌重塑的研究进展

心肌重塑是心脏在一些生理的或病理的刺激作用下,心肌细胞和心肌细胞外基质在细胞结构、功能、数量及遗传表型方面出现的明显的变化即心脏的大小、形状和功能的变化。心肌细胞和心肌细胞外基质从根本上参与了心肌重塑的过程。目前,对于影响心肌重塑的因素及作用机制的研究主要集中在血流动力学和神经体液方面。近年来,对于不良心肌重塑的逆转干预,包括药理干预、运动干预,一直持续不断,研究的不断深入给相关疾病的改善、治疗带了新的进展和希望。心肌重塑可能是生理性的或病理性的,生理性的重塑是心肌的适应性代偿性变化,而病理性的重塑是心肌的不适应变化,对身体产生危害性。本文主要对病理性心肌重塑的主要组成部分,影响心肌重塑的因素及相关机制,改善不良心肌重塑的有效干预做一个综述,并提出展望。     

人表皮生长因子的研究进展

人表皮生长因子是激活表皮生长因子受体的生长因子家族的典型成员,由人体的多个组织器官合成与分泌,通过结合受体激活一系列信号途径,调控细胞的增殖、分化和迁移等。近年来,有关人表皮生长因子的研究已扩展到其在人类生理和病理作用的领域,尤其在组织再生和伤口愈合方面成为研究热点。文中综述了人表皮生长因子的研究进展,简要描述了其基因和蛋白的结构与特点、作用机制与生物学效应,重点介绍该生长因子在胃肠溃疡愈合、皮肤伤口修复和肿瘤病理过程中的作用与影响,从而为相关研究提供辅助信息。     

表皮生长因子与哺乳动物生殖的关系

表皮生长因子与哺乳动物生殖的关系岳占碰杨增明（东北农业大学生物工程系哈尔滨１５００３０）关键词表皮生长因子哺乳动物生殖表皮生长因子（ｅｐｉｄｅｒｍａｌｇｒｏｗｔｈｆａｃｔｏｒ,ＥＧＦ）是一种多肽类生长因子,该因子在人和动物各种组织器官生长发育过程中是...     

A role of heparin-binding epidermal growth factor-like growth factor in cardiac remodeling after myocardial infarction

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is known to induce cell growth in various cell types via transactivation of epidermal growth factor receptor (EGFR). To investigate the involvement of HB-EGF and EGFR in cardiac remodeling after myocardial infarction (MI), we examined the expressions of mRNA and protein in rat hearts 6 weeks after MI-induction. Where increased expressions of HB-EGF mRNA and protein were observed, infarcted myocardium was replaced by extracellular matrix and interstitial fibroblasts. EGFR mRNA and protein expression did not show significant changes in sham-operated heart tissues, non-infarcted region, and infarcted region. In vitro study demonstrated that HB-EGF mRNA was expressed mainly in cultured fibroblasts rather than in myocytes. We suggest that the interaction between HB-EGF and EGFR transactivation is closely related to the proliferation of cardiac fibroblasts and cardiac remodeling after MI in an autocrine, paracrine, and juxtacrine manner.     

Heparin-Binding EGF-Like Growth Factor Induces Heart Interstitial Fibrosis via an Akt/mTor/p70s6k Pathway

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is essential for maintaining normal function of the adult heart and is known to play an important role in myocardial remodeling. In the present study, we observed that heart-specific HB-EGF transgenic (TG) mice had systolic dysfunction with decreased fractional shortening (FS%), increased end-systolic diameter (LVIDs) at 5 months of age, increased heart fibrosis, and increased mRNA expression of Col1α1 and Col3α1 at 1, 3, 5 and 7 months of age compared to nontransgenic (NTG) littermates. However, the left ventricular anterior wall thickness at end-systole (LVAWs) of the TG mice was not different than the NTG mice. Phosphorylation levels of Akt, mTor and p70s6k were increased due to HB-EGF expression in TG mice compared with the NTG mice at 3 and 7 months of age. Additionally, activated Akt, mTor and p70s6k were co-localized with vimentin to cardiac fibroblasts isolated from TG mice. Furthermore, HB-EGF significantly increased phosphorylation levels of Akt, mTor and p70s6k and increased expression of type I collagen in cultured primary cardiac fibroblasts. Rapamycin (Rapa) and CRM197, inhibitors of mTor and HB-EGF respectively, could inhibit the expression of type I collagen in the cultured primary cardiac fibroblasts and Rapa suppressed interstitial fibrosis of the heart tissues in vivo. In addition, a BrdU assay showed that HB-EGF increased proliferation of cardiac fibroblasts by 30% compared with cells without HB-EGF treatment. HB-EGF-induced proliferation was completely diminished in the presence of Rapa. These results suggest that HB-EGF induced heart fibrosis and proliferation of cardiac fibroblasts occurs through activation of the Akt/mTor/p70s6k pathway.     

A Short Form of the Heparin-binding EGF-like Growth Factor with a Changed EGF Domain

In all secreted proteins related to the epidermal growth factor (EGF), EGF domains that occur in a mature factor are each encoded by two exons, and those that do not, by one exon. During splicing, additional exon 3a can be inserted between exons 3 and 4, which code for the EGF domain of the mature heparin-binding EGF-like growth factor (HB-EGF). The resulting mRNA codes for the short form of HB-EGF (SF HB-EGF), which retains the signal peptide, the propeptide, and the heparin-binding domain. However, its EGF domain lacks the C-terminal subdomain essential for the interaction with the EGF receptor (EGFR). Structural analysis suggested that SF HB-EGF is a secreted polypeptide that has high affinity for heparin but weakly, if at all, interacts with EGFR. Data obtained in three different systems indicated that SF HB-EGF possesses a mitogenic activity but utilizes a signal transduction pathway other than that of HB-EGF.     

High-Level Expression and Purification of Heparin-Binding Epidermal Growth Factor (HB-EGF) with SUMO Fusion

Heparin-binding epidermal growth factor (HB-EGF) can stimulate the division of various cell types and has potential clinical applications that stimulate growth and differentiation. HB-EGF has an EGF-like domain typical of all members of the EGF family. The high expression of active HB-EGF in Escherichia coli has not been successful as the protein contains three intra-molecular disulfide bonds, the same as other members of the EGF super family that are difficult to form correctly in the bacterial intracellular environment. This work fused the non-glycosylated HB-EGF gene with a small ubiquitin-related modifier gene (SUMO) by over-lap PCR. The resulting fusion gene SUMO-HBEGF was highly expressed in BL21(DE3) that the soluble SUMO-HBEGF was up to 30% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease Ulp1 to obtain the native HB-EGF, which was further purified by Ni-NTA affinity chromatography. MTT assays indicated the purified HB-EGF, as well as SUMO-HBEGF, had mitogenic activity in a dose-dependent manner.     

Structure of heparin-binding EGF-like growth factor. Multiple forms, primary structure, and glycosylation of the mature protein.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a newly described member of the epidermal growth factor (EGF) family that is mitogenic for BALB/c 3T3 cells, inhibits the binding of 125I-EGF to its receptor, and triggers autophosphorylation of the EGF receptor. HB-EGF was purified from the conditioned medium of U-937 cells using cation exchange, copper affinity, heparin affinity, and two rounds of C4 reversed phase liquid chromatography. The elution profile of the first round of C4 column chromatography contained four growth factor activity peaks with similar specific biological activities. N-terminal and tryptic fragment microsequencing demonstrated that these peaks contained different structural forms of the HB-EGF protein. Some of the differences in the various forms of HB-EGF were found to be due to N-terminal heterogeneity. Microsequencing of tryptic fragments indicated that the mature HB-EGF polypeptide can contain at least 86 of the 208 amino acids predicted by nucleotide sequence to be the HB-EGF precursor molecule. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the various forms of HB-EGF have apparent molecular masses of 19-23 kDa. Further analysis of the most predominant form of HB-EGF found in U-937 cell conditioned medium indicated that it has a pI of 7.2-7.8 and is O-glycosylated.     

Generation and Characterization of Conditional Heparin-Binding EGF-Like Growth Factor Knockout Mice

Recently, neurotrophic factors and cytokines have been shown to be associated in psychiatric disorders, such as schizophrenia, bipolar disorder, and depression. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family, serves as a neurotrophic molecular and plays a significant role in the brain. We generated mice in which HB-EGF activity is disrupted specifically in the ventral forebrain. These knockout mice showed (a) behavioral abnormalities similar to those described in psychiatric disorders, which were ameliorated by typical or atypical antipsychotics, (b) altered dopamine and serotonin levels in the brain, (c) decreases in spine density in neurons of the prefrontal cortex, (d) reductions in the protein levels of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor and post-synaptic protein-95 (PSD-95), (e) decreases in the EGF receptor, and in the calcium/calmodulin-dependent protein kinase II (CaMK II) signal cascade. These results suggest the alterations affecting HB-EGF signaling could comprise a contributing factor in psychiatric disorder.     

Roles of Dppa2 in the regulation of the present status and future of pluripotent stem cells

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a membrane-anchored protein, known as proHB-EGF. ProHB-EGF is cleaved by metalloproteases through a process referred to as 'ectodomain shedding', resulting in the formation of soluble HB-EGF. Both proHB-EGF and soluble HB-EGF are biologically active; the former acts on neighbouring cells through juxtacrine signalling, whereas the latter can move to distant locations. Elevated HB-EGF expression has been observed in ovarian and some other cancers. CRM197, a diphtheria toxin (DT) mutant, binds directly to the epidermal growth factor (EGF)-like domain and represses the mitogenic activity of HB-EGF. Recently, monoclonal antibodies (mAbs) specific for human HB-EGF were generated by immunizing HB-EGF-deficient mice with human HB-EGF (Hamaoka et al. (2010) J. Biochem. 148, 55-69). Most of the mAbs can bind to the EGF-like domain of HB-EGF, but fail to inhibit the mitogenic activity of soluble HB-EGF. However, some mAbs prevented the ectodomain shedding of proHB-EGF and inhibited the proliferation of EGF receptor-expressing cells stimulated by proHB-EGF-expressing cells. Hamaoka et al. showed that CRM197 prevents the ectodomain shedding of proHB-EGF. Thus, these mAbs function as specific inhibitors for the ectodomain shedding of HB-EGF and may be useful for treating cancers exhibiting elevated levels of HB-EGF.