肝细胞生长因子在胃癌病人血清中的表达及意义

目的:肝细胞生长因子(hepatocyte growth factor,HGF)是一种多肽生长因子,其促进细胞增殖,诱导细胞迁移、侵袭及参与血管生成,在肿瘤发生发展过程中具有重要作用。本研究通过检测胃癌病人血清HGF水平,分析其与胃癌临床病理特征的关系,并与常用肿瘤标志物CA19-9、CEA进行比较,探讨胃癌病人血清中HGF的表达及其临床意义。方法:用酶联免疫吸附夹心法检测80例胃癌病人术前血清HGF、CA19-9、CEA水平,并观察60例病人根治术后血清HGF变化。结果:胃癌病人血清HGF水平(2.94±2.67)μg/L明显高于正常对照组(1.02±0.31)μg/L(P=0.002);分层分析发现,随着疾病的进展,血清HGF水平逐渐升高[Ⅰ期(2.19±2.12)μg/L,Ⅱ期(2.53±2.38)μg/L,Ⅲ期(3.92±3.45)μg/L,Ⅳ期(4.13±3.71)μg/L];Ⅲ期和Ⅳ期分别与Ⅰ期进行比较,均有显著差异(P<0.05)。另外,80例病人血清HGF、CA19-9和CEA的阳性率分别为80%、16.2%和5.0%,HGF阳性率明显较高(P<0.01)。结论:胃癌病人血清HGF水平明显高于正常对照组,且血清HGF的表达水平与胃癌淋巴结及肝脏转移呈正相关.血清HGF表达水平可作为反映胃癌生物学行为和判断预后的参考指标。     

Overexpression and regulation of expression of scatter factor/hepatocyte growth factor in prostatic carcinoma

Objectives. Scatter factor (hepatocyte growth factor) (SF/HGF) is a multifunctional polypeptide growth factor that has been implicated in tumor proliferation, angiogenesis, invasiveness, and metastasis. Little is known of the expression of SF/HGF in human prostatic carcinoma. The aims of this investigation were to quantitate the level of SF/HGF expression in benign versus malignant human prostatic tissues and to assess regulation of SF/HGF expression by human prostatic stromal myofibroblasts.Methods. We determined the level of SF/HGF expression in 10 human prostatic tissue samples (5 benign, 5 carcinoma) by Western blot analysis. Five purified growth factors—basic fibroblast growth factor (bFGF), interleukin-1beta (IL-1β), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and endothelial growth factor (EGF)—were tested for their capacity to induce SF/HGF expression by a human prostatic stromal myofibroblastic cell line, as assessed by enzyme-linked immunosorbent assay. Supernatant from the normal PrEC prostatic epithelial cell line and the DU 145 carcinoma cell line were assayed for SF/HGF-inducing activity.Results. SF/HGF exhibited a mean fourfold overexpression in carcinoma tissues compared with benign prostatic tissue. Significant stimulation of SF/HGF expression by prostatic stromal myofibroblasts was detected for IL-1β (8.1-fold), PDGF (6.2-fold), bFGF (4.0-fold), VEGF (3.7-fold), and EGF (2.9-fold). DU 145-conditioned media, but not the PrEC-conditioned media, contained SF/HGF-inducing activity, which was determined to include IL-1β, bFGF, and PDGF by antibody-blocking experiments.Conclusions. SF/HGF is overexpressed in human prostatic carcinoma tissues. Prostatic carcinoma cell stimulation of SF/HGF expression by adjacent benign myofibroblastic cells as a type of epithelial–stromal paracrine interaction could potentially influence prostatic carcinoma cell behaviors.     

胃癌组织中E钙黏素基因启动子甲基化状态的研究

目的 研究胃癌组织中E钙黏素(E-CD)基因启动子区甲基化的状况.方法 采用甲基化特异性PCR方法,检测106例胃癌组织及其邻近小凹上皮中E-CD基因启动子区甲基化状态.结果 在慢性胃炎小凹上皮、胃癌邻近小凹上皮及胃癌组织中E-CD基因启动子区甲基化率分别为0(0/15),22.6%(25/106)和50.0%(53/106),三者之间差异均有统计学意义(P＜0.01).Ming浸润型胃癌E-CD基因启动子区甲基化率(62.7%,37/59)和Laurén弥漫型胃癌E-CD基因启动子区甲基化率(64.5%,40/62)分别明显高于Ming膨胀型胃癌甲基化率(34.0%,16/47)和Laurén肠型胃癌E-CD基因启动子区甲基化率(29.5%,13/44),二者之间差异均有统计学意义(P＜0.01).甲基化与胃癌的分化程度、浸润深度、区域淋巴结转移和pTNM分期亦有关(P＜0.05).结论 E-CD基因启动子区甲基化在胃癌组织中普遍存在,它可能是胃癌发生的早期事件.胃癌E-CD基因启动子区甲基化状态与胃癌生物学行为密切相关.     

Influence of hepatocyte growth factor secreted from fibroblasts on the growth and invasion of scirrhous gastric cancer

The objective of this study was to show that hepatocyte growth factor (HGF) and HGF receptor (c-met protein) play an important role in the cancer growth and infiltration in scirrhous gastric cancer. The expression level of c-met protein was examined in 90 cases of advanced gastric cancer using anti-c-met antibody. Co-cultivation of each of four gastric cancer cell lines with gastric fibroblasts was performed using a double chamber method. The expression rate of c-met was 79.5% in type 4 tumors, significantly higher than in other types. The expression rates were 63.6% in undifferentiated-type cancer and 36.3% in differentiated-type cancer. Co-cultivation of undifferentiated-type cancer with fibroblasts showed a significantly higher HGF concentration than fibroblasts cultured alone. The growth in three undifferentiated-type cancers was accelerated by an addition of rhHGF and by co-cultivation with fibroblasts and was inhibited by anti-HGF antibody. Moreover rhHGF stimulated the invasion activity of undifferentiated cancer cell lines. These findings suggested that gastric fibroblasts in scirrhous cancer stimulate tumor growth and invasion through activation of the HGF/c-met system.     

转染HGF基因对肝细胞的生物学效应

目的探讨肝细胞生长因子(hepatocyte growth factor，HGF)基因对肝细胞生长增殖能力的影响。方法通过脂质体介导法，将HGF基因导入肝细胞中。用荧光显微镜以及原位杂交观察到HGF基因表达。采用检测细胞生长曲线、Ki-67蛋白和嗜银蛋白免疫组化观察肝细胞生长增殖能力及DNA合成能力的变化。结果荧光显微镜观察可见到绿色荧光蛋白的表达，用原位杂交方法进一步证实了HGF蛋白在细胞中的表达。细胞生长曲线显示，转染HGF基因的肝细胞增殖速度明显增快，Ki-67蛋白和嗜银蛋白表达明显增多，提示转导HGF基因使肝细胞增殖活性增加。结论本实验显示转染的HGF可表达并有促细胞分裂活性。为进一步了解HGF分子生物学特性及治疗应用提供了理论基础。     

转肝细胞生长因子基因肝细胞模型的建立

目的：利用脂质体介导法在体外建立转肝细胞生长因子（HGF）基因的人肝细胞模型。方法：建立HGF真核细胞表达载体，利用脂质体介导法在体外将HGF基因转染入人肝细胞，利用荧光显微镜观察、免疫组织化学、原位杂交方法检测HGF真核细胞表达载体的转录和表达情况。结果：以阳离子脂质体LipofectAMINE为载体将HGF基因转染人肝细胞后，经400mg/L的G418筛选后可形成抗性克隆；Neo基因原位杂交结果显示转染基因的细胞有阳性表达；荧光显微镜下观察到有绿色荧光；免疫组织化学证实转染HGF基因的肝细胞有HGF蛋白的表达。结论：HGF基因可被成功转染入人肝细胞并能有效表达，这可能为肝病的基因治疗提供一种新途径。     

肝细胞生长因子及其受体在膀胱癌中的表达及意义

目的探讨肝细胞生长因子及其受体c-Met在人膀胱移行细胞癌中的表达及临床意义。方法采用免疫组化方法检测8例正常膀胱黏膜标本及45例膀胱移行细胞癌标本中HGF及c-Met的表达;并以分级、分期、复发、转移等临床特征分组,比较各组的阳性表达率。结果8例正常膀胱黏膜中HGF均为阴性表达,c-Met仅1例为弱阳性表达。而45例膀胱移行细胞癌标本中,两者阳性表达率分别为64.4%和62.2%,共表达率42.2%。肿瘤组织与正常膀胱黏膜比较,HGF及c-Met的表达差异均有统计学意义。肿瘤初发和复发组HGF阳性表达率差异有统计学意义(P<0.01);Tis~T1组及T2~T4组c-Met的阳性表达率差异有统计学意义(P<0.01)。结论HGF及其受体c-Met在膀胱移行细胞癌中呈过度表达,该信号传导通路主要以自分泌方式激活,在膀胱移行细胞癌的发生、发展及术后复发中可能起重要作用。     

Inhibition of DNA synthesis by somatostatin in rat hepatocytes stimulated by hepatocyte growth factor or epidermal growth factor.

The antiproliferative effects of somatostatin on hepatocytes stimulated by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) were investigated using primary cultures of adult rat hepatocytes. Somatostatin inhibits HGF-induced (at a dose of 10 ng/mL) or EGF-induced (at a dose of 100 ng/mL) 3H-thymidine incorporation into hepatocytes in a dose-dependent manner (10(-10) to 10(-8) M). This inhibition was confirmed by autoradiography. The effect of somatostatin was nontoxic as judged by preserved albumin synthesis, a marker for differentiated hepatocyte function. In the presence or absence of somatostatin, neither HGF nor EGF significantly altered intracellular cyclic adenosine monophosphate (cAMP). We conclude that somatostatin is a potent inhibitor of HGF- or EGF-induced deoxyribonucleic acid synthesis in adult rat hepatocytes. The mechanism of this inhibition appears to be independent of cAMP. The significance of somatostatin in liver regeneration has yet to be assessed.     

血管内皮生长因子D促进胃癌淋巴管生长

目的研究血管内皮生长因子D（VEGF-D）与胃癌淋巴管转移之间的关系。方法采用反转录-聚合酶链反应（RT-PCR）从人胃癌旁正常组织中扩增人VEGF-D全长cDNA，经T-A克隆酶切鉴定及测序得到完整的序列后，亚克隆并构建pEGFP／VEGF-D真核表达载体，转染低表达VEGF-D的胃癌细胞株SGC7901，通过体外G418抗生素筛选得到高表达VEGF-D的肿瘤细胞克隆，用这种高表达VEGF-D的胃癌细胞接种于裸鼠皮下，用淋巴管内皮细胞透明质酸盐受体（LYVE-1）行免疫组织化学染色观察肿瘤淋巴管密度和形态。结果对照组和转染pEGFP／VEGF-D组肿瘤的重量分别为（1．13±0．40）g和（2．24±0．82）g，两组差异有统计学意义（P〈0．05）。肿瘤组织内的淋巴管密度分别为2．89±1．32和5．74±1．30，两组差异有统计学意义（P〈0．01）。周边肿瘤组织存在扩张的功能性淋巴管。结论VEGF-D在胃癌中可能通过主动促进淋巴管生长来促进肿瘤生长和远处转移。     

肝细胞生长因子与胃癌淋巴管生成及淋巴转移的关系

目的明确肝细胞生长因子（HGF）与胃癌淋巴管生成的相互关系及其可能途径，进一步探索HGF在肿瘤淋巴转移中的作用。方法对60例胃癌组织及其邻近正常胃组织和20例胃溃疡组织标本作免疫组织化学染色，并记数血管内皮生长因子-C（VEGF-C）、HGF、c-Met和淋巴管内皮透明质酸受体-1（Lyve-1），结合胃癌的临床病理资料作相关统计学分析。结果胃癌组织中VEGF-C、HGF、c-Met表达明显上调，其微淋巴管密度（MLD）也明显高于正常胃组织和胃溃疡组织（P〈0．001），有无淋巴转移和远处转移差异有统计学意义（P〈0．05）；胃癌和胃溃疡边缘淋巴管内皮有c-Met表达，HGF和肿瘤MLD之间有明显的正相关系（P〈0．001）和多元线性回归关系（P=0．001）。结论HGF与VEGF．C一样是胃癌的淋巴管生成刺激因子，它可以通过直接或间接途径刺激淋巴管的增生，促进肿瘤淋巴转移。     

The role of hepatocyte growth factor in growth plate cartilage

To investigate the physiological role of hepatocyte growth factor (HGF) in endochondral bone formation, we examined the expression of HGF and its receptor c-met and the effects of HGF on growth plate chondrocytes. HGF was highly expressed in the prehypertrophic zone and hypertrophic zone in rat costal growth plate cartilage. The expression of HGF increased in rabbit chondrocytes as they matured in culture. Conversely, c-met expression was down regulated along maturation of growth plate chondrocytes. HGF had weak stimulatory effects on DNA and proteoglycan synthesis of growth plate chondrocytes. However, HGF strongly inhibited expression of terminal differentiation-related phenotypes, such as type X collagen and alkaline phosphatase (APase) synthesis and cartilage matrix mineralization. When HGF was removed from the cultures, cells quickly expressed type X collagen and APase. Once chondrocytes differentiated to mature chondrocytes, HGF did not inhibit further differentiation of these cells. These results suggested that HGF is a negative regulator of terminal differentiation of growth plate chondrocytes.. Received: Feb. 12, 1998 / Accepted: March 12, 1998     

肝细胞生长因子诱导骨髓间充质干细胞向肝细胞分化的实验研究

目的探讨成年大鼠骨髓间充质干细胞(MSCs)在体外是否可定向诱导分化为肝细胞及其方法。方法采用梯度离心法,分离纯化SD大鼠的MSCs,流式细胞仪检测和碱性磷酸酶染色鉴定细胞类型。根据培养基中肝细胞生长因子(HGF)浓度不同,将MSCs分为4组进行诱导分化：A组0 ng／ml,B组10ng／ml,C组20ng／ml,D组40ng／ml。倒置显微镜连续观察细胞分化过程的形态学变化。于培养的1,3,7,14,21,28 d,分别以逆转录聚合酶链反应(RT-PCR)和细胞免疫组化检测各组细胞甲胎蛋白(AFP)、细胞角蛋白18(CK18)和白蛋白的基因和蛋白表达。结果分离纯化的SD大鼠MSCs的表面标志为CD29^ ,CD44^ ,CD34^-,CD45^-和CD90^ ,细胞的碱性磷酸酶染色阴性,细胞纯度达98％以上。C组与D组的MSCs,于培养第7天出现AFP基因表达,第14天表达增强,第28天表达减弱;第14天始出现白蛋白、CK18基因表达,而后持续。B组和A组在培养过程中,未出现AFP、CK18和白蛋白的表达。C组与D组MSCs的AFP细胞免疫组化染色培养第7天即出现阳性,第14天白蛋白和CK18免疫组化染色阳性。A组和B组的MSCs的AFP、白蛋白和CK18的免疫组化染色均阴性。结论成年大鼠MSCs在较高浓度的HGF的诱导下,可分化为肝细胞。     

Promotion of rabbit ligament healing by local delivery of hepatocyte growth factor

Background  

Extracapsular ligament injuries of the knee and ankle are common injuries. Ligaments heal slowly, usually over months or longer by scar formation rather than by tissue regeneration. This study was performed to evaluate the therapeutic effect of locally delivered recombinant hepatocyte growth factor (HGF) on the early healing of ligaments in a rabbit model.     

Regulation of connective tissue growth factor (CTGF) by hepatocyte growth factor in human tubular epithelial cells

BACKGROUND: Hepatocyte growth factor (HGF) is a pleiotropic protein with renoprotective functions, which have been attributed at least in part to its ability to counteract the profibrotic effects of transforming growth factor beta (TGF-beta). A major downstream mediator of TGF-beta is connective tissue growth factor (CTGF). However, the molecular mechanisms of CTGF regulation by HGF have not yet been investigated. METHODS: CTGF expression was analysed in human primary tubular epithelial cells (hPTECs) and the cell line HKC-8 by western blotting. Morphological alterations were analysed by immunocytochemistry. RESULTS: HGF induced a transient expression of CTGF, which was maximal after 6 h and returned to baseline after 24 h. Coincubation with TGF-beta increased CTGF protein at 6 h, whereas HGF significantly decreased CTGF induction by TGF-beta after 24 h. Furthermore, HGF induced cell scattering associated with reorganization of focal adhesions and formation of lamellipodia and filopodia. The early induction of CTGF was linked to the HGF-mediated alterations of cell morphology. The PP2 inhibitor of Src-family kinases, which regulate focal adhesion turnover, reduced HGF-mediated upregulation of CTGF. In addition, inhibition of the Rho-kinase, which modulates the actin cytoskeleton, impaired CTGF expression. Combination of both inhibitors further decreased CTGF expression. Comparable inhibitory effects were obtained, when CTGF was induced by the combination of HGF and TGF-beta. CONCLUSIONS: We provide evidence for a dual effect of HGF on CTGF regulation in human tubular epithelial cells: transient upregulation of CTGF in the absence of TGF-beta, which was related to alterations of cell morphology, and interference with TGF-beta-mediated CTGF induction after prolonged incubation.     

肝细胞生长因子受体在高糖诱导肾I间质成纤维细胞中的表达及其意义

目的体外观察高糖刺激成纤维细胞时,肝细胞生长因子(HGF)受体c-met的表达及HGF/c-met系统的活性.方法高糖刺激细胞后,采用RT-PCR方法分析HGF、c-met mRNA水平.Western印迹分析c-met蛋白质.同时检测外源加入HGF对TGF-β、c-met表达影响,以及采用抗c-met抗体处理后的表达.结果高糖刺激早期HGF、c-met mRNA快速上升.另外,c-met mRNA、蛋白水平明显上调.HGF可以诱导c-met表达,同时降低TGF-β表达.结论c-met局部上调在糖尿病肾脏损伤过程中起重要作用.     

替米沙坦上调肝细胞生长因子表达的分子机制研究

目的探讨替米沙坦对肝细胞生长因子（hepatocyte growth factor, HGF）表达的影响及其机制研究。方法选取体外培养大鼠肾系膜细胞,观察不同浓度（0．1μmol／L、1．0μmol／L、10．0μmol／L）替米沙坦干预24h对HGF表达的影响;采用双荧光素酶报告基因分析系统检测HGF启动子活性和替米沙坦的PPAR7反应性。非放射性蛋白激酶检测系统及Westernblot检测PKA、CREB、pCREB蛋白表达。结果与正常对照组相比,替米沙坦能明显增加HGF蛋白表达,该作用呈浓度依赖性（P〈0．05）;荧光素酶检测证实HGF基因的-290／＋20序列存在潜在PPAR7反应元件,10μmol／L替米沙坦显著增强HGF启动子活性（P〈0．05）。同时蛋白检测提示替米沙坦抑制系膜细胞PKA活性,下调pCREB蛋白表达。结论替米沙坦上调HGF表达独立于PKA／CREB信号通路,可能通过结合HGF启动子PPAR7反应元件上调HGF表达。     

Sustained induction of hepatocyte growth factor by sensitization with freeze-thawed hepatic tissue

Hepatocyte growth factor (HGF) is a mitogen for hepatocytes, and has therapeutic potential for fibrotic/cirrhotic liver. Therefore, the induction of HGF in vivo is considered to be useful in the treatment of liver dysfunction caused by cirrhosis, chronic hepatitis, or extensive surgical resection. In this study, we examined the sustained induction of HGF by inoculation of freeze-thawed hepatic tissue (FTHT). Serum from rats inoculated with FTHT increased [³H]thymidine incorporation i.e., increased DNA synthesis, in primary cultured rat hepatocytes. The DNA synthesis was significantly promoted by the addition of the FTHT-sensitized serum, while this DNA synthesis was inhibited by neutralizing anti-rat HGF antibody. The concentration of HGF in the FTHT-sensitized serum was increased by day 3 after the inoculation. The time of HGF induction was dependent on the inoculated volume of FTHT, but peaks of HGF concentration were found on day 5 with different volumes of FTHT. Injurins, inducers of HGF, were also induced in the FTHT-sensitized rats, with their peak levels on day 3. The FTHT inoculated tissue showed inflammatory cell infiltration, which was gradually absorbed, and had completely disappeared by day 14 after the inoculation. Although mild inflammatory cell infiltration was observed in non-freeze-thawed inoculated hepatic tissue (NFHT) a tight capsule formed around the NFHT, and was scarcely phagocytized on day 14. These results suggest that FTHT inoculation induces HGF sustainedly through the increased synthesis of injurins, and that freeze-thawed tissue, which is easily phagocytized, is important for the sustained induction of HGF. Received for publication on Sept. 6, 1997; accepted on April 8, 1998     

肝细胞生长因子受体在高糖诱导肾间质成纤维细胞中的表达及其意义

目的 体外观察高糖刺激成纤维细胞时，肝细胞生长因子（HGF）受体c-met的表达及HGF／c-met系统的活性。方法 高糖刺激细胞后，采用RT－PCR方法分析HGF、c-met mRNA水平。Western印迹分析c-met蛋白质。同时检测外源加入HGF对TGF－β、c-met表达影响，以及采用抗c-met抗体处理后的表达。结果 高糖刺激早期HGF、c-met mRNA快速上升。另外，c-met mRNA、蛋白水平明显上调。HGF可以诱导c-met表达，同时降低TGF－β表达。结论 c-met局部上调在糖尿病肾脏损伤过程中起重要作用。