不同浓度肝细胞生长因子和成纤维细胞生长因子 对大鼠肝干细胞的增殖调控

目的：初步探讨不同浓度的肝细胞生长因子(hepatocyte growth factor,HGF)和成纤维细胞生长因子(fibroblast growth factor, FGF)对大鼠肝干细胞增殖的调控作用。方法：先将大鼠肝胎细胞分别种植A、B培养板上进行肝细胞的体外培养。A组：培养液中含有HGF,浓度分为别1,5,10,20,40,80及100 ng/mL。B组：培养液中含有FGF,浓度分别为1,5,10,20,40,80及100 ng/mL。对照组不加细胞因子。由A、B两组剂量效应获得HGF和FGF的最佳浓度。再将细胞接种于C组培养板进行培养,培养液中含有最佳浓度HGF联合上述各浓度FGF, 并用四甲基偶氮唑盐比色法(mononuclear cell direct cytotoxicity assay, MTT)分别测两组的光密度值（optical delnsity, OD）,检测出HGF与FGF的最佳浓度组合。结果：HGF和FGF浓度与肝干细胞增殖效应呈剂量依赖性（dose-dependent）。当HGF浓度为20 ng/mL,FGF浓度为10 ng/mL时,为最佳浓度组合。结论：HGF和FGF均能对肝干细胞的增殖起促进作用,并且两者有一定协同作用。     

Plasminogen-mediated activation and release of hepatocyte growth factor from extracellular matrix

Interventions that enhance plasminogen activation within the lung consistently limit the fibrosis that follows alveolar injury. However, this protective effect cannot be attributed solely to accelerated clearance of fibrin that forms as a provisional matrix after lung injury. To explore other mechanisms, we considered interactions between the plasminogen activation system and hepatocyte growth factor (HGF). HGF is known to have antifibrotic activity, but to do so, it must be both released from its sites of sequestration within extracellular matrix (ECM) and activated by proteolytic cleavage. A recent study using bleomycin-exposed mice showed that manipulations of the plasminogen activation system influenced the amount of free HGF within bronchoalveolar lavage fluid without affecting total lung HGF mRNA or protein. To elucidate the mechanisms, we studied the role of plasminogen activation in fibroblast-mediated HGF release and activation. We found that NIH3T3 and mouse lung fibroblasts release ECM-bound HGF in a plasminogen-dependent fashion. The plasminogen effect was lost when lung fibroblasts from urokinase-type plasminogen activator (uPA)-deficient mice were used, and was increased by fibroblasts from plasminogen activator inhibitor (PAI)-1-deficient mice. Plasminogen addition to NIH3T3 or mouse lung fibroblasts increased conversion of pro-HGF to its active form. The plasminogen effect on activation was lost when uPA-deficient fibroblasts were used and accentuated by PAI-1-deficient fibroblasts. In conjunction with the previous in vivo study, these results suggest that plasminogen activation can protect the lung against fibrosis by increasing the availability of active HGF.     

肝细胞生长因子与妊娠期高血压疾病

肝细胞生长因子(hepatocyte growth factor,HGF)是一种具有多效性作用的肝素结合性酸性蛋白.HGF在胎盘中主要由绒毛核心间质细胞表达,旁分泌作用于邻近滋养细胞表面受体c-met.妊娠期高血压疾病时,HGFmRNA及蛋白产物表达下降.HGF在调节滋养细胞浸润能力、抗细胞凋亡和胎盘发生方面与妊娠期高血压疾病关系密切.     

Regulation of spreading and growth of colon cancer cells by hepatocyte growth factor

Hepatocyte growth factor (HGF), also known as scatter factor, regulates both cell motility and the growth of some cell types. We have determined the effects of HGF on the motility and growth of human colon cancer cell lines (HT115, HT29, HRT18 and HT55). Cell motility, as measured by dissociation from carrier beads or by scattering of cell colonies, was greatly increased in all cell lines. The effects were completely blocked by anti-HGF antibody. In contrast, cell growth of HT115, HT29 and HRT18 cells was inhibited by a wide range of concentrations of HGF. HT55 cell growth was also inhibited but needed a prolonged culture period (>5 days). The HGF receptor/Met protein is highly expressed in the membrane fraction of these cells as determined by Western blotting. It is concluded that HGF has an effect on both colon cancer cell motility and growth, which may be important in the control of the spread of colon cancer.     

Estrogen and progesterone receptor expression in macrophages and regulation of hepatocyte growth factor by ovarian steroids in women with endometriosis

BACKGROUND: Information regarding macrophage-mediated regulation of hepatocyte growth factor (HGF) by ovarian steroid hormones in women with endometriosis is limited. Therefore, we investigated the regulation of HGF by steroid hormones in isolated macrophages and stromal cells derived from women with or without endometriosis. METHODS: We isolated CD68 immunoreactive adherent macrophages in vitro from 46 women with endometriosis and 30 women without endometriosis. Estrogen receptor (ER) and progesterone receptor (PR) expression in macrophages was demonstrated by immunohistochemistry and RT-PCR. Production of HGF in the culture media of basal and ovarian steroid-stimulated macrophages was examined by enzyme-linked immunosorbent assay. Expression of mRNA for HGF and its receptor, c-Met in macrophages and stromal cells in response to ovarian steroid was investigated by RT-PCR. The single and combined effect of HGF and estrogen on the growth of macrophages and stromal cells was analysed by bromodeoxyuridine (BrdU) incorporation. RESULTS: ER and PR were expressed in isolated macrophages and intact tissue at the protein and mRNA levels. Macrophages derived from women with endometriosis produced significantly higher concentration of HGF (352.2 +/- 4.9 pg/ml) in conditioned media after treatment with estradiol (10(-8) mol/l) than that of basal macrophages (221.5 +/- 32.8 pg/ml, P<0.05) or women without endometriosis (170.6 +/- 2.6 pg/ml, P<0.05). These effects were less evident after treatment with progesterone. Treatment with tamoxifen (10(-6) mol/l) reversed the production of HGF and other macromolecules. Secretion of HGF in response to ovarian steroids was further enhanced after activation with lipopolysaccharide. The mRNA expressions of HGF and its receptor, c-Met, were also detected in macrophages and stroma in response to estrogen, suggesting an autocrine regulation. HGF mRNA expression was higher in cells of women with endometriosis than non-endometriosis women. Bromodeoxyuridine incorporation indicated that exogenous stimulation with HGF and estrogen, either alone or in combination, significantly increased the cell proliferation of both endometrial stroma and macrophages compared to that of non-endometriosis or non-treated cells. CONCLUSION: These results suggest that besides other inflammatory mediators, ovarian steroids also participate in the production of HGF by peritoneal macrophages which may be involved in the growth of endometriosis either alone or in combination with estrogen.     

转染肝细胞生长因子基因对CCl4损伤人肝细胞株的保护作用

我们已观察到转染肝细胞生长因子(hepatocyte growth factor,HGF)基因对培养肝细胞DNA台成具有显著的刺激作用。但是转染HGF基因对肝细胞是否具有直接的细胞保护作用尚未见报道。本实验利用培养的人肝细胞株QZG,观察转染肝细胞生长因子对四氯化碳(carbon tetrachloride,CCl4)染毒肝细胞的保护作用。     

Anti-allergic inflammatory effects of hepatocyte growth factor

Hepatocyte growth factor (HGF) is known to influence a number of cell types and regulate various biological activities including cytokine production, cell migration, proliferation and survival. Thus, HGF is now recognized to be a key factor in the prevention and attenuation of disease progression. We have reported that HGF reduces allergic airway inflammation, airway hyperresponsiveness, remodeling and development of Th2 cytokines as well as growth factors such as transforming growth factor-beta in vivo. In vitro, HGF directly attenuates chemotaxis of eosinophils in the absence of Th2 cytokines and modulates mitogen-activated protein kinases, which play an important role in eosinophil migration. In this review, we discuss the physiological role of HGF in allergic inflammation and its mechanism of anti-inflammatory effects, including the regulation of eosinophil functions.     

Modulation of growth factor action by the extracellular matrix

Growth factors were historically defined as molecules produced by the body to regulate cell growth and proliferation. After the identification of their receptors and the intracellular signaling machinery they activate, it is now clear that they are involved in the regulation of multiple processes essential for development and normal tissue function. The present review gives a brief overview of growth factor action. Receptor activation and signaling are discussed, highlighting the role of extracellular matrix interactions.     

肝细胞生长因子和表皮生长因子联合体外诱导大鼠肝卵圆细胞分化的研究

目的探求卵圆细胞向成熟肝细胞分化的演变规律,并对其分化调控的分子机制作初步探讨。方法联合应用肝细胞生长因子和表皮生长因子体外培养大鼠肝脏卵圆细胞OC3,运用电镜、免疫细胞化学、半定量逆转录-聚合酶链反应（RT—PCR）等技术,检测OC3在分化过程中形态及相关分子标记物的改变,并运用蛋白芯片技术观察细胞蛋白表达谱的变化。结果诱导10周后,卵圆细胞胞质变丰富,细胞器增多,表达谷胱甘肽-S-转移酶（GST—P）和丙酮酸激酶（M2-PK）减弱,而表达白蛋白（ALB）、CK18增强,同时诱导后期的细胞较之诱导前主要出现了8种表达差异蛋白。结论在肝细胞生长因子与表皮生长因子的共同作用下,体外培养的卵圆细胞可逐步分化为成熟肝细胞。8种主要的结构蛋白或功能蛋白可能参与了对该过程的调控。     

肝细胞生长因子和类胰岛素样生长因子对心肌干细胞的诱导作用

目的探讨肝细胞生长因子(HGF)和类胰岛素样生长因子(IGF1)在外能否诱导心肌干细胞(CSCs)发生增殖并向心肌细胞定向分化。方法组织贴块法分离培养心肌干细胞,免疫荧光技术鉴定c-kit和CD34表达,流式细胞仪分选纯化c-kit+细胞,CFDA SE荧光探针示踪培养检测细胞增殖特征。实验分为单纯心肌干细胞组和与心肌共培养组,分别用HGF和IGF1干预,倒置显微镜观察细胞不同时期数量与形态的变化,活细胞工作站观察CFDA SE示踪剂荧光强弱,采集图像,进行统计学分析。免疫荧光技术检测Nkx2.5、心肌肌钙蛋白T(cTnT)的表达。结果单纯心肌干细胞组,生长因子刺激后心肌干细胞数量与形态均无明显变化。与心肌共培养组,细胞均发生增殖与形态变化,Nkx2.5、cTnT阳性表达,有个别心肌干细胞分化为自发搏动的心肌细胞。结论心肌干细胞与心肌细胞共培养条件下,HGF和IGF1能够促进心肌干细胞增殖,联合作用能够诱导心肌干细胞向心肌细胞分化。     

Neutrophil priming by hepatocyte growth factor, a novel cytokine.

We demonstrate here that the recently defined cytokine hepatocyte growth factor (HGF) 'primes' human neutrophils. Recombinant human HGF over the concentration range 0.1-20 ng/ml increased the neutrophil response to f-met-leu-phe by up to 200%, and required only a short preincubation, 10 min producing the maximum effect. Priming was independent of changes in cytosolic-free calcium homeostasis. We conclude that HGF may be a physiologically important cytokine with 'priming' activity for neutrophils.     

Activation of hepatocyte growth factor by the plasminogen activators uPA and tPA.

Hepatocyte growth factor, also known as scatter factor, is a complete mitogen for hepatocytes that bears sequence and structural homology with plasminogen. Because it exists in both a mitogenically inactive single-chain form and an active two-chain form, we were interested in determining whether plasminogen activators could properly cleave single-chain hepatocyte growth factor to generate active two-chain hepatocyte growth factor. Herein we report that both urokinase-type plasminogen activator and tissue-type plasminogen activator can cleave single-chain hepatocyte growth factor, generating two-chain hepatocyte growth factor. When equal quantities of plasminogen activator-treated and activator-untreated hepatocyte growth factor are compared in serum-free in vitro bioassays, the treated hepatocyte growth factor is mitotically more active. Also, urokinase-type plasminogen activator was inactive against hepatocyte growth factor molecules with a mutated cleavage site. This suggests that urokinase-type and tissue-type plasminogen activator may be natural biological regulators of hepatocyte growth factor. Because the active form of hepatocyte growth factor is a powerful stimulator of DNA synthesis and cell motility, these findings may be relevant in understanding the role of plasminogen activators in the biology of cancer invasion and metastasis.     

Effect of hepatocyte growth factor on sperm motility

PROBLEM: Hepatocyte growth factor (HGF) exists abundantly in seminal plasma and its receptor, c-met, is expressed on spermatozoa. Considering its motogenic activity, we speculated that HGF might affect the movement ability of spermatozoa. METHODS: Recombinant HGF was added to washed spermatozoa and their movements were analyzed using a computer-assisted sperm analyzer. The concentration of HGF in the seminal plasma of infertile patients (n = 83) was measured by ELISA, and the data were compared with their hormonal profile and semen parameters. RESULTS: The HGF physiological concentration (1 ng/mL) maintained the motility of sperm after a long incubation, though the difference was not statistically significant. Recombinant HGF did not affect the linearity or frequency of movement, which suggested that it does not evoke the hyperactivation of spermatozoa. The concentration of HGF in seminal plasma did not correlate with any clinical parameter of the patients. CONCLUSIONS: These findings contradict the theory that HGF controls the movement of sperm. The main role of this axis in the male reproductive system might be maturation in the epididymis.     

Molecular recognition and modulation of hepatocyte growth factor activity by heparan and dermatan sulfates

Introduction Hepatocyte growth factor/scatter factor (HGF/SF) is an unusual growth factor in that it binds both heparan sulfate (HS) ( Lyon et al. 1994 ) and dermatan sulfate (DS) ( Lyon et al. 1998 ) glycosaminoglycans (GAGs) with similar high affinities. Both these GAGs act as co‐receptors for HGF/SF in the activation of the Met receptor ( Lyon et al. 2002 ). Our aim was to determine the sequences in HS and DS that specifically interact with and modulate HGF/SF activity. Materials and methods A structurally unique DS, which possesses O‐sulfation at carbon‐6 of the hexosamine residue (and not carbon‐4 as in mammalian DS), was obtained from the sea cucumber, Ascidia nigra. A variety of HS‐ and DS‐like structures were also generated using various chemical modification procedures (specific desulfations and carboxyl reductions). The ability of these various GAG species to compete with cell surface GAGs for HGF/SF binding was tested using radiolabelled HGF/SF and MDCK cells. The modified GAG structures and the A. nigra DS are currently being tested for their ability to act as co‐receptors for the interaction between HGF/SF and Met by studying cell signalling and cellular response assays, using the sulfated GAG‐deficient CHO‐745 cell line. Results Unexpectedly, A. nigra DS was found to bind HGF/SF strongly with a KD of around 1 nm . This interaction is 20‐fold stronger than that of between HGF/SF and mammalian DS, but similar to that of with HS. A. nigra DS also stimulated HGF/SF‐mediated Erk activation and migration in CHO‐745 cells. Studies using the modified GAG species showed that, in the case of HS, 6‐O‐sulfate and N‐sulfate groups are most important for HGF/SF binding. For HGF/SF binding to DS, hexosamine O‐sulfate is most important. HGF/SF was also found to bind 6‐O‐sulfated GAGs more strongly than 4‐O‐sulfated ones. Discussion The data show that there is flexibility in the structures recognized by HGF/SF, and this explains the ability of the growth factor to bind both HS and DS. However, there are still observable preferences in GAG structure, such as 6‐O‐sulfation over 4‐O‐sulfation. Information on HGF/SF‐binding GAG structures is valuable for the design of HGF/SF antagonists that could be useful therapeutically in the treatment of solid tumours where HGF/SF‐Met activity is up‐regulated.     

肝细胞生长因子在缺血性疾病的研究及进展

肝细胞生长因子(hepatocyte growth factor,HGF)是一类具有多功能生物活性的生长因子,参与机体多种组织器官及细胞的生长、再生、重塑、促血管新生等过程。因其具有生物活性的多样性,被广泛用于基础实验及临床研究,然而其对血管内皮细胞的增生及维护内皮细胞功能从而促进血管新生加速其侧枝循环作用,被广泛用于缺血性疾病的研究,本文就近年来HGF在缺血性疾病等方面进行综述。     

Distinct regulation of myoblast differentiation by intracellular and extracellular fibroblast growth factor-1

We studied the role of fibroblast growth factor (FGF)-1 in the physiology of myoblast differentiation. We found that, while endogenous FGF-1 in L6-10 rat myoblasts did not suppress the progress of differentiation, the addition of FGF-1 to the culture medium suppressed it. Moreover, L6-10 cells stably transfected with full length FGF-1 undergo enhanced differentiation. The latter was well correlated with myogenin expression and myotube formation. Constitutive expression of a mutant FGF-1 (FGF-1U) that lacked a nuclear localization signal, promoted the differentiation of the myoblasts even more strongly. Furthermore, the expression of FGF-1U in an inducible expression system enhanced myogenin expression promptly. In L6-10 transfectants expressing a dominant-negative mutant of FGF receptor, stable transfection of FGF-1 promoted differentiation as it did in parent cells. Studies with FGF receptors and MAP kinase suggest that both are involved in the effect of FGF-1 when it is supplemented to culture medium but not during the effect of endogenous FGF-1 synthesized in cells. We conclude that intracellular (endogenous) and extracellular (exogenous) FGF-1 have differential effects on the regulation of myogenic differentiation of L6-10 cells.     

腺病毒介导的肝细胞生长因子/血管内皮生长因子双基因转染骨髓间充质干细胞移植治疗心肌梗死***

背景：骨髓间充质干细胞可以分化为心肌细胞,促进血管再生,但在移植早期其自身分泌的细胞因子不足以维持良好的分化和再生。 目的：验证腺病毒介导的肝细胞生长因子和血管内皮生长因子双基因转染新西兰兔骨髓间充质干细胞移植对新西兰兔梗死心肌组织的修复重建和血管再生的影响。 方法：腺病毒介导肝细胞生长因子/血管内皮生长因子双基因转染BrdU标记的新西兰兔骨髓间充质干细胞。取新西兰兔50只建立急性心肌梗死模型,4周后随机分为5组,分别于梗死心肌内注射：①骨髓间充质干细胞/Ad.血管内皮生长因子+肝细胞生长因子。②骨髓间充质干细胞/Ad. 肝细胞生长因子。③骨髓间充质干细胞/Ad.血管内皮生长因子。④骨髓间充质干细胞。⑤对照组注射等量无血清IMDM培养液。移植4周后观察移植细胞的分化和新生血管的形成,并通过超声多普勒检测心功能变化。 结果与结论：除对照组外,其余4组兔心功能都较移植前有明显改善(P < 0.05),其中移植双基因转染骨髓间充质干细胞/Ad.血管内皮生长因子+肝细胞生长因子组兔的心功能改善程度要明显高于其他3组。部分BrdU染色阳性的细胞可以分化成为内皮细胞,参与构成了梗死区域的新生毛细血管。与对照组比较,其余4组都有明显的血管新生(P < 0.05),而以骨髓间充质干细胞/Ad.血管内皮生长因子+肝细胞生长因子组最显著。提示肝细胞生长因子/血管内皮生长因子双基因转染新西兰兔骨髓间充质干细胞移植于梗死心肌可以促进心肌再生和新生血管的形成,明显改善心功能。 关键词：基因转染;骨髓间充质干细胞;血管内皮生长因子;肝细胞生长因子;移植 doi:10.3969/j.issn.1673-8225.2012.10.029