角质细胞生长因子研究进展

角质细胞生长因子（KGF）属于成纤维细胞生长因子家族,它通过与受体（KGFR）的结合,特异性地刺激上皮细胞的增殖.KGF基因表达受到正负调控作用,正负调控作用的平衡对于KGF正常发挥功能具有重要意义.KGF具有多种生物学功能：参与组织、器官的发育,具有损伤防治功能,与癌症的发生有着密切的联系.     

角质细胞生长因子(KGF)研究进展

角质细胞生长因子(KGF)属于成纤维细胞生长因子(FGFs)家族的成员之一,又称FGF一7,最早由Rubin等(1980)从人类胎肺成纤维细胞培养液中分离提纯获得的。人KGF的cDNA编码一含194个氨基酸的单链多肽,其分子量为26-28KD。KGF由各种来源的间质细胞分泌,与肝素有较强的亲和力,其受体KGFR属于蛋白酪氨酸激酶受体家族,目前已知该家族主要包括FGFR1、FGFR2、FGFR3和FGFR4四位成员,KGFR由FGFR2基因编码,为FGFR-2的剪接形式,即FGFR2Ⅲb,其主要分布于上皮细胞,KGF与靶细胞膜上的受体KGFR特异性结合后,促使受体自身磷酸化,从而启动细胞内信号级联反应,进而发挥多种生物学功能:参与组织器官发育、促进细胞增殖及组织损伤修复、减少放化疗引起的副反应,尤其与癌症的发生发展有着密切的联系。该文就KGF的研究进展进行综述。     

角质细胞生长因子的研究进展

角质细胞生长因子（KGF）是成纤维细胞生长因子（FGFs）家族的成员,即FGF－7,最初是从人胚胎肺成纤维细胞的培养上清中分离纯化获得的。成熟KGF为一163个氨基酸残基的单链多肽,分子量为26－28KD。KGF由各种来源的间质细胞分泌,受体分布于上皮细胞,其生物学活性是特异性地促进上皮细胞的增殖、迁移和分化。FGF的表达受激素和一些细胞因子的调控。有关研究表明,KGF对肺泡Ⅱ型细胞的增殖以及皮肤、胃肠道粘膜和角膜损伤的修复具有十分重要的作用。     

角质形成细胞生长因子-2的应用研究进展

角质形成细胞生长因子-2（KGF-2）是成纤维细胞生长因子（FGFs）超家族中角质细胞生长因子家族的一员。KGF-2能够促进上皮细胞的增殖、分化和迁移,参与并调控脊椎动物多种组织和器官的形成。本文就其生物学功能,以及在疾病治疗和临床试验等方面的研究进展进行综述。     

角质细胞生长因子的研究进展

角质细胞生长因子(KGF)是成纤维细胞生长因子(FGFs)家族的成员,即FGF-7,最初是从人胚胎肺成纤维细胞的培养上清中分离纯化获得的。成熟KGF为一163个氨基酸残基的单链多肽,分子量为26—28KD。KGF由各种来源的间质细胞分泌,受体分布于上皮细胞,其生物学活性是特异性地促进上皮细胞的增殖、迁移和分化。KGF的表达受激素和一些细胞因子的调控。有关研究表明,KGF对肺泡Ⅱ型细胞的增殖以及皮肤、胃肠道粘膜和角膜损伤的修复具有十分重要的作用。     

角质细胞生长因子-2研究进展

角质细胞生长因子 2 (KGF 2 )也叫成纤维细胞生长因子 10 (FGF 10 ) ,是成纤维细胞生长因子家族的一员 .能特异性促进上皮细胞的增殖、分化和迁移 ,对脊椎动物多种组织和器官的发育起重要调控作用 ,对临床上多种疾病的治疗也有很好的应用前景 .1　KGF 2与受体KGF 2有两种细胞膜表面受体 :FGFR1Ⅲb和FGFR2Ⅲb .KGF 2与FGFR2Ⅲb的亲和力很高 ,而与FGFR1Ⅲb的亲和力很低 ,只有在高浓度KGF 2存在时才与FGFR1Ⅲb结合 .KGF 2与受体结合后 ,促使受体胞内的C端酪氨酸残基磷酸化 ,磷酸化的受体具有了酪氨酸蛋白激酶活性 ,并与一系…     

人角质细胞生长因子2基因的克隆及其在大肠杆菌中的表达

目的:构建人角质细胞生长因子2(hKGF2)基因的高效原核表达载体pET-30a( )-hKGF2,并在大肠杆菌BL21(DE3)中获得表达。方法:从培养的人胚胎肺成纤维细胞中提取总RNA,采用RT-PCR技术扩增出去除了信号肽部分的hKGF2基因,克隆到pMD18-T载体,经DNA序列分析后与pET-30a( )表达载体连接,在大肠杆菌BL21(DE3)诱导表达6×His-hKGF2,用SDS-PAGE及Western印迹鉴定表达蛋白。结果:构建了表达载体pET-30a( )-hKGF2,经IPTG诱导后,表达的重组蛋白理论相对分子质量约为23000,约占菌体总蛋白的20%。结论:6×His-hKGF2蛋白在大肠杆菌BL21(DE3)中为可溶性高效表达,为获得高纯度、高活性的产物,以及进一步的大规模生产和应用研究奠定了基础。     

人角质细胞生长因子2真核表达载体的构建及功能验证

目的:构建人角质细胞生长因子2(KGF-2)基因真核表达载体,探索KGF-2的上皮细胞迁移功能。方法:以人乳腺cDNA文库为模板,PCR扩增KGF-2基因片段,将其插入pXJ-40-myc,经双酶切和测序验证后,将重组质粒转染HEK-293T细胞,采用Western印迹检测重组蛋白;转染人肠上皮细胞FHC,采用细胞划痕实验检测细胞迁移能力。结果:PCR扩增获得627 bp的DNA产物,插入pXJ-40-myc载体,双酶切及测序结果证明重组质粒含有目的序列;转染HEK-293T细胞,Western印迹检测到相对分子质量约23×103的目的蛋白;细胞划痕实验显示,转染Myc-KGF-2的FHC细胞较未转染或转染空载体的细胞迁移能力强。结论:构建了人KGF-2基因真核表达载体,并验证了其促进细胞迁移的功能。     

人角质细胞生长因子2在不同原核表达系统中的表达差异

目的:比较重组人角质细胞生长因子2(rhKGF-2)在不同原核表达系统中的表达量和表达稳定性之间的差异。方法:利用PCR方法从人胚胎肺成纤维细胞cDNA扩增得到hKGF-2序列,双酶切后分别克隆到pBV220、pQE31和pET-24b载体中,分别转化大肠杆菌JM109、M15和BL21(DE3)进行诱导表达,SDS-PAGE分析rhKGF-2的表达量和表达稳定性,并纯化rhKGF-2。结果:pBV220-rhKGF-2与pET-24b-rhKGF-2在宿主菌内经诱导后均有目的蛋白表达,其中pBV220-rhKGF-2的表达量约占菌体总蛋白的10%,pET-24b-rhKGF-2的表达量约占菌体总蛋白的25%,且均为可溶性表达,但后者的表达稳定性明显优于前者,而pQE31-rhKGF-2在宿主菌内几乎没有表达。结论:hKGF-2在不同原核表达系统中的表达量和表达稳定性存在明显差异。     

Protein engineering,expression, and activity of a novel fusion protein possessing keratinocyte growth factor 2 and fibronectin

Growthh factor-induced proliferation and differentiation often require adhesion of cells to the extracellular matriv proteins such as fibronectin （FN）. in this study, we aimed to investigate the effect of protein engineering of the keratinocyte growth factor 2 （KGF2） fused to the FN on the mitogenic activity of KGF2. The fusion pratein （KGF2-FN10）, which was expressed in Escherichia coil, showed significantly enhanced mitogenie activity of KGF2 on human keratinocytes. Moreover, KGF2-FN10 fusion protein showed significantly increased activity to differentiate keratinocytes from native KGF2. In conclusion, these results suggest that KGF2-FN10 fusion protein has certain advantages over native KGF2 and may offer a novel strategy to potentiate the therapeutic effect of KGF2.     

Cortactin involvement in the keratinocyte growth factor and fibroblast growth factor 10 promotion of migration and cortical actin assembly in human keratinocytes

Keratinocyte growth factor (KGF/FGF7) and fibroblast growth factor 10 (FGF10/KGF2) regulate keratinocyte proliferation and differentiation by binding to the tyrosine kinase KGF receptor (KGFR). KGF induces keratinocyte motility and cytoskeletal rearrangement, whereas a direct role of FGF10 on keratinocyte migration is not clearly established. Here we analyzed the motogenic activity of FGF10 and KGF on human keratinocytes. Migration assays and immunofluorescence of actin cytoskeleton revealed that FGF10 is less efficient than KGF in promoting migration and exerts a delayed effect in inducing lamellipodia and ruffles formation. Both growth factors promoted phosphorylation and subsequent membrane translocation of cortactin, an F-actin binding protein involved in cell migration; however, FGF10-induced cortactin phosphorylation was reduced, more transient and delayed with respect to that promoted by KGF. Cortactin phosphorylation induced by both growth factors was Src-dependent, while its membrane translocation and cell migration were blocked by either Src and PI3K inhibitors, suggesting that both pathways are involved in KGF- and FGF10-dependent motility. Furthermore, siRNA-mediated downregulation of cortactin inhibited KGF- and FGF10-induced migration. These results indicate that cortactin is involved in keratinocyte migration promoted by both KGF and FGF10.     

病毒dUTPase研究进展

脱氧尿苷焦磷酸酶（dUTP pyrophosphatase,dUTPasc）广泛存在于真核、原核细胞和病毒等生物有机体中,通过催化水解脱氧尿苷三磷酸（dUTP）,减少尿嘧啶在DNA合成中的错误掺入,降低细胞中的dUTP／dTTP比例,保证DNA复制的正确性和顺利进行。病毒编码的dUTPasc具有种属特异性,且与病毒的毒力和高效复制密切相关。本文就dUTPase的生物学功能、分类特征、表达调控、分布定位及病毒dUTPase功能的研究进展进行了概述,为深入开展dUTPase功能研究提供理论基础。     

HaCaT keratinocyte migration is dependent on epidermal growth factor receptor signaling and glycogen synthase kinase-3alpha

After epithelial disruption by tissue injury, keratinocytes migrate from the wound edge into a provisional matrix. This process is stimulated by growth factors that signal through epidermal growth factor (EGF) receptor, including EGF, heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor-alpha (TGF-alpha), and by for example keratinocyte growth factor (KGF) and TGF-beta1 that function through different receptors. We have previously shown that keratinocyte migration induced by EGF or staurosporine is dependent on the activity of glycogen synthase kinase-3 (GSK-3). In the present study, we show that keratinocyte migration induced by TGF-beta1, KGF, EGF, TGF-alpha and staurosporine depends on EGFR signaling, involves autocrine HB-EGF expression and is potently blocked by GSK-3 inhibitors SB-415286 and LiCl. Inhibition of GSK-3 also retards wound reepithelialization in vivo in mice. Moreover, inhibition of GSK-3 activity prevented cell rounding that is an early event in EGFR-mediated keratinocyte migration. Isoform-specific GSK-3alpha and GSK-3beta knockdown and overexpression experiments with siRNAs and adenoviral constructs, respectively, revealed that GSK-3alpha is required for keratinocyte migration, whereas excessive activity of GSK-3beta is inhibitory. Thus, induction of keratinocyte migration is conveyed through EGFR, promoted by endogenous HB-EGF and requires GSK-3alpha activity.     

Probing the structure-function relationship of nerve growth factor

We compared the receptor binding, antigenicity, biological activation, and cell-mediated proteolytic degradation properties of mouse nerve growth factor (mNGF) and human NGF (hNGF). The affinity of hNGF toward human NGF-receptor is greater than that of mNGF, but the affinity of mNGF toward rat NGF-receptor is greater than that of hNGF. Thus, the specificity of the interaction between NGF and its receptor resides both on the NGF and on its receptor. Using a group of anti-NGF monoclonal antibodies that competitively inhibit the binding of NGF to receptor, sites differing between mNGF and hNGF were detected. Together, these results indicate that the sites on hNGF and mNGF, responsible for binding to NGF-receptor, are similar but not identical. In comparing the relative abilities of mNGF and hNGF to stimulate a biological response in PC12 cells, we observed that mNGF was better at stimulating neurite outgrowth than was hNGF, consistent with the differences observed for receptor binding affinity. However, the ED₅₀ for biological activation is approximately 100-fold lower than theK_d for receptor occupancy, and, thus, the dose-response curve is not consistent with a simple activation proportional to receptor occupancy. The data are consistent with a model requiring a low-level threshold occupancy of NGF-receptor (K_d=10^–9 M) in order to stimulate full biological activity. Finally, we observed the degradation of NGF by PC12 cells. We found that the NGF molecule is significantly degraded via a receptor-mediated uptake mechanism. Together, the data provide insight into regions of the NGF molecule involved in contacts with the receptor leading to formation of the NGF: NGF-receptor complex. Additionally, they establish the link between occupancy of receptor and biological activation and the requirement for receptor-mediated uptake in order to degrade NGF proteolytically in cultured PC12 cells.     

Ligand-induced clathrin-mediated endocytosis of the keratinocyte growth factor receptor occurs independently of either phosphorylation or recruitment of eps15

Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells. Following ligand binding, KGFR is rapidly activated and internalized by clathrin-mediated endocytosis. Among the possible receptor substrates which could be involved in the regulation of KGFR endocytosis and down-modulation, we analyzed here the eps15 protein in view of the proposed general role of eps15 in regulating clathrin-mediated endocytosis as well as that of eps15 tyrosine phosphorylation in the control of regulated endocytosis. Immunoprecipitation and Western blot analysis showed that activated KGFR was not able to phosphorylate eps15, suggesting that eps15 is not a receptor substrate. Double immunofluorescence and confocal microscopy revealed that activated KGFR, differently from epidermal growth factor receptor (EGFR), did not induce recruitment of eps15 to the cell plasma membrane. Microinjection of a monoclonal antibody directed against the C-terminal DPF domain which contains the AP2 binding region of eps15 led to inhibition of both pathways of receptor-mediated endocytosis, the EGFR ligand-induced endocytosis and the transferrin constitutive endocytosis, but did not appear to block the KGFR ligand-induced internalization. Taken together our results indicate that the clathrin-mediated uptake of KGFR is not mediated by eps15.