血管内皮生长因子及其受体在多发鼻息肉中的表达

多发鼻息肉的治疗和预后与其生长部位和形态特点有密切关系，其中大部分为起源部位不连续的多个单发息肉，一般有蒂或广基，好发部位为窦口鼻道复合体、额隐窝。但有一小部分多发鼻息肉为双侧鼻腔、鼻窦黏膜广泛炎症反应和息肉样变性，息肉、息肉样变黏膜与正常黏膜无明显分界线，鼻腔、鼻窦黏膜弥漫性水肿样增生，无蒂，无好发部位可寻，中鼻甲甚至鼻中隔也发生同样病变，复发率相当高。本实验以这两组不同类型多发鼻息肉为研究对象，就血管内皮生长因子（vascular endothelial growth factor，VEGF）及其受体血管内皮生长因子受体2（vascular endothelial growth factor receptor2，VEGFR2）在其中的表达情况进行研究和分析。

Real-time Quantitative PCR Analysis of Vascular Endothelial Growth Factor Receptor-2(VEGFR-2) Expression at Zebrafish Different Developmental Stages

[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2（VEGFR-2） at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish embryos and larvae.Real-time quantitative RT-PCR was performed to examine the expression of VEGFR-2.The data were analyzed by 2^-△△Ct method.[Result]The expression level of VEGFR-2 gene increased gradually from 12 to 72 hpf,and subsequently decreased at 96 hpf.The expression level was lowest at 12 hpf,highest at 72 hpf,and had significant differences when compared with that of other developmental stages.[Conclusion]The expression level of VEGFR-2 increases gradually before blood vessel maturation and decreases as blood vessels mature.

生长因子受体连接蛋白-2在膀胱移行上皮癌诊断中的应用价值

目的 探讨成纤维细胞生长因子受体连接蛋白-2（FGFB2）表达与膀胱移行细胞癌发生、进展程度的关系.方法 用免疫组织化学和Westem blot法检测64例膀胱移行细胞癌标本中FGFR2的表达情况,并与正常胱膀组织中表达情况相比较,观察FGFR2表达与病理分级、肿瘤浸润程度及治疗过程的关系.结果 在病理分级和FGFR2阳性表达之间有相反趋势,FGFR2阳性表达率随着病理分级的增加而降低,FCFR2在Ⅰ～Ⅲ级、对照组问表达差异有统计学意义（P=0.03）;在浸润组（T2-T4）中FGFR2阳性表达明显低于表浅组（Tis-T1）（P=0.004）;在复发组FGFR2阳性表达显著低于未复发组（P=0.042）.结论 FGFR2表达量与膀胱移行细胞癌的疾病严重程度呈相反趋势,与复发有关.

Expression of thymidine kinase mediated by a novel non-viral delivery system under the control of vascular endothelial growth factor receptor 2 promoter selectively kills human umbilical vein endothelial cells

AIM: To investigate the killing efficiency of a recombinant plasmid containing a thymidine kinase (TK) domain insert driven by the vascular endothelial growth factor receptor 2 (VEGFR2) promoter (KDR) on vascular endothelial cells.METHODS: The KDR-TK fragment was extracted from pBluescript Ⅱ KDR-TK plasmid by enzymatic digestion with Xho I and Sal I. The enhanced green fluorescence protein (EGFP) carrier was extracted from pEGFP by the same procedure. The KDR-TK was inserted into the pEGFP carrier to construct pEGFP-KDR-TK. Using ultrasound irradiation and microbubble, pEGFP-KDR-TK was transferred into human umbilical vein endothelial cells (HUVECs). The transient infection rate was estimated by green fluorescent protein (GFP) expression. Transfected HUVECs, non-transfected HUVECs, and HepG2 cells were cultured in the presence of different concentrations of ganciclovir (GCV), and the killing efficacy of HSV-TK/GCV was analyzed by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: The recombinant pEGFP-KDR-TK was successfully constructed by inserting the KDR-TK fragment into the pEGFP carrier. Transfected HUVECs showed cytoplasmic green fluorescence, and the transient transfection rate was about 20.3%. Pools of G418-resistant cells exhibited a higher sensitivity to theprodrug/GCV compared to non-transfected HUVECs or non-transfected HepG2 cells, respectively. CONCLUSION: KDR promoter and the suicide gene/prodrug system mediated by diagnostic ultrasound combined with microbubble can significantly kill HUVECs. Such therapy may present a novel and attractive approach to target gene therapy on tumor vessels.

Lysophosphatidic acid transactivates both c-Met and epidermal growth factor receptor, and induces cyclooxygenase-2 expression in human colon cancer LoVo cells

AIM： To examine whether lysophosphatidic acid （LPA） induces phosphorylation of c-Met and epidermal growth factor receptor （EGFR）, both of which have been proposed as prognostic markers of colorectal cancer, and whether LPA induces cyclooxygenase-2 （COX-2） expression in human colon cancer cells. METHODS： Using a human colon cancer cell line, LoVo cells, we performed immunoprecipitation analysis, followed by Western blot analysis. We also examined whether LPA induced COX-2 expression, by Western blot analysis. RESULTS： Immunoprecipitation analysis revealed that 10 μmol/L LPA induced tyrosine phosphorylation of c-Met and EGFR in LoVo cells within a few minutes. We found that c-Met tyrosine phosphorylation induced by LPA was not attenuated by pertussis toxin or a matrix metalloproteinase inhibitor, in marked contrast to the results for EGFR. In addition, 0.2-40 IJmol/L LPA induced COX-2 expression in a dose-dependent manner. CONCLUSION： Our results suggest that LPA acts upstream of various receptor tyrosine kinases （RTKs） and COX-2, and thus may act as a potent stimulator of colorectal cancer. 2005 The WJG Press and Elsevier Inc. All rights reserved.

成骨细胞特异性因子2periostin及相关因子在大鼠全层缺损皮肤创面愈合过程中的表达

目的观察大鼠皮肤缺损创面愈合过程中成骨细胞特异性[大J子2periostin（PN）与血管生成素-1（Ang-1）、血管内皮生长因子（VEGF）及其受体2[VEGFR-2／胎肝激酶-1（FLK-1）]在创面及创缘周围皮肤的表达及对创面愈合的影响。方法48只成年雄性SD大鼠按随机数字表法分成6组，每组8只。在大鼠背中部脊柱两侧各制作一个2μm×2cm全层皮肤缺损创面，分别于术后1、4、7、10、14、21d创面取材，行病理组纵学检查及免疫组化染色检测创面PN、Ang-1、VEGF、FLK-1的表达；并以正常皮肤的表达量作为对照组。结果创面局部组织中各闵子的表达量在术后均迅速升高。与对照组比较，创面PN的表达量在术后1d即增加了234．4％，之后持续升高，至7d增加了597．9％并达到峰值，其后缓慢下降，21d时增长率为280．9％，仍维持在较高水平（均P〈0．05）；Ang-1的表达量在术后1d增加了128．1％，4d增加了327．5％并达到峰值，然后逐渐下降，14d的增长率仅为80．5％，之后轻度升高（均P〈0．05）；VEGF的表达量在术后7d增加了165．8％并达到峰值，而后下降并伴有小幅波动（均P〈0．0S）；FLK-1术后1d增加了56．1％并维持这一水平，至7d增加了70．1％达峰值（均P〈0．05），10d后缓慢下降（均P〉0．05）。结论大鼠皮肤创面愈合过程中，PN、Ang-1、VEGF及其受体FLK-1蛋白均高表达，但各因子的表达高峰和持续时间有所不同。PN的表达量增幅最高且始终维持较高水平至创面表皮化后；说明PN、Ang-1、VEGF、FLK-1共同参与了大鼠皮肤创面的愈合，PN存创面愈合的整个过程中发挥着重要作用。

Inhibition of tumor angiogenesis by TTF1 from extract of herbal medicine

AIM:To study the inhibition of tumor angiogenesis by 5,2,4'-trihydroxy-6,7,5'-trimethoxyflavone(TTF1) isolated from an extract of herbal medicine Sorbaria sorbifolia.METHODS:Angiogenic activity was assayed using the chick embryo chorioallantoic membrane(CAM) method.Microvessel density(MVD) was determined by staining tissue sections immunohistochemically for CD34 using the Weidner capillary counting method.The mRNA and protein levels of vascular endothelial growth factor(VEGF),vascular endothelialgrowth factor receptor 2(VEGFR2,Flk-1/KDR),basic fibroblast growth factor(bFGF),cyclo-oxygenase(COX)-2 and hypoxia-inducible factor(HIF)-1α were detected by quantitative real-time polymerase chain reaction and Western blotting analysis.RESULTS:The TTF1 inhibition rates for CAM were 30.8%,38.2% and 47.5% with treatment concentrations of 25,50 and 100 μg/embryo × 5 d,respectively.The inhibitory rates for tumor size were 43.8%,49.4% and 59.6% at TTF1 treatment concentrations of 5,10,and 20 μmol/kg,respectively.The average MVD was 14.2,11.2 and 8.5 at treatment concentrations of 5 μmol/kg,10 μmol/kg and 20 μmol/kg TTF1,respectively.The mRNA and protein levels of VEGF,KDR,bFGF,COX-2 and HIF-1α in mice treated with TTF1 were significantly decreased.CONCLUSION:TTF1 can inhibit tumor angiogenesis,and the mechanism may be associated with the down-regulation of VEGF,KDR,bFGF,HIF-1α and COX-2.

肝内胆管癌中FGFR2突变的分子机制及诊断和治疗现状

肝内胆管癌是一种高度恶性且易复发的肝脏肿瘤。手术、化疗和放疗等治疗手段对肝内胆管癌的疗效较差。测序和组学技术的进步为肝内胆管癌的靶向治疗提供了方向。成纤维细胞生长因子受体2(fibroblast growth factor 2,FGFR2)突变作为肝内胆管癌中好发的基因异常之一,其分子机制及致癌能力已通过基础研究得到了较为深入的解析,针对带有FGFR2突变的肝内胆管癌的靶向治疗也已经应用于临床并取得了良好的治疗效果。本文从基础研究和临床策略2方面出发,系统地回顾了肝内胆管癌中FGFR2突变致癌的具体分子机制和携带FGFR2突变的肝内胆管癌的临床诊疗现状。

Screening of miRNAs associated with lymph node metastasis in Her-2-positive breast cancer and their relationship with prognosis

The aim of this study was to identify some biomarkers for predicting lymph node metastasis and prognosis of human epidermal growth factor receptor 2(Her-2)-positive breast cancer(BC). We analyzed correlations between micro RNAs(mi RNAs) and the prognosis of patients with BC based on data collected from The Cancer Genome Atlas(TCGA) database. The expression levels of mi R-455, mi R-143, and mi R-99 a were measured in clinical samples of Her-2-positive BC patients with different degrees of lymph node metastasis. We investigated the impacts of overexpressed mi R-455 on the proliferation and invasiveness of MDA-MB-453 cells and measured its effects on the expression of long non-coding RNA(lnc RNA) metastasis-associated lung adenocarcinoma transcript 1(MALAT1) by quantitative real-time polymerase chain reaction(q RT-PCR). The expression of mi R-455 was significantly and positively correlated to the prognosis and overall survival(OS) of the BC(P=0.028), according to TCGA information. The expression level of mi R-455 was positively correlated with OS and relapse-free survival(RFS) of patients with Her-2-positive BC, and was negatively correlated with the number of metastatic lymph nodes(P<0.05). Transwell assay suggested that MDA-MB-453 cells became much less invasive(P<0.01) after being transfected with mi R-455 mimics. During the q RT-PCR, the expression level of MALAT1 declined significantly after transfection(P<0.01). Overexpressed mi R-455 significantly inhibited the proliferation and migration of MDA-MB-453 cells and the expression of MALAT1. We conclude that mi R-455 may be a useful potential biomarker for forecasting lymph node metastasis and the prognosis of Her-2-positive BC patients. mi R-455 may play an important role in lymph node metastasis of BC by interacting with MALAT1.

HER-2 overexpression and survival in colorectal cancer: a meta-analysis

Objective： Numerous studies examining the relationship between human epidermal growth factor receptor 2 （HER-2） overexpression and survival in patients with colorectal cancer （CRC） have yielded controversial results. We therefore performed a meta-analysis more precisely to estimate its prognostic value. Methods： Published studies investigating the effect of HER-2 overexpression on CRC survival were identified; the hazard ratios （HRs） and their corresponding 95% confidence intervals （95% Cls） were pooled in terms of disease-specific or overall survival. Results Eleven studies were included in the meta-analysis. The pooled data showed that HER-2 overexpression was negatively related to CRC survival （HR=1.10, 95% CI： 0.77-1.44）. Subgroup analyses regarding test method and study quality also demonstrated little association between HER-2 overexpression and CRC survival （HR=0.89, 95% CI： 0.50-1.29; HR=0.90, 95% Ch 0.43-1.37, respectively）, Conclusions： Regardless of several limitations, our study suggested that HER-2 overexpression probably had little impact on CRC survival.

Overweight and obesity versus concentrations of VEGF-A, sVEGFR-1, and sVEGFR-2 in plasma of patients with lower limb chronic ischemia

Objective： Being overweight or obese comprises a significant risk factor for atherosclerosis. Fat tissue also generates factors stimulating angiogenesis, the process by which new blood vessels form. The purpose of this paper is to assess concentrations of the vascular endothelial growth factor A （VEGF-A） and its soluble type-1 and type-2 re- ceptors （sVEGFR-1 and sVEGFR-2） in plasma of patients with peripheral arterial disease （PAD） depending on the level of nutrition according to body mass index （BMI）. Methods： The study group included patients suffering from symptomatic PAD （n=46） in Fontaine classes Ila-lV without any history of neoplastic disease and who have a normal BMI （n=15）, are overweight （n=21） or are obese （n=10）. The control group （n=30） consisted of healthy non-smoking volunteers who were neither overweight nor obese. Venous blood plasma samples were collected from both groups at rest in the morning to determine plasma concentrations of VEGF-A, sVEGFR-1, and sVEGFR-2 using the enzyme- linked immunosorbent assay （ELISA） method. Results： The group of patients with PAD co-existent with being over- weight or obese tended to have higher mean concentration levels of VEGF-A and sVEGFR-2 when compared with patients suffering from PAD with normal BMIo A statistically significant positive correlation was obtained between BMI and average plasma concentrations of sVEGFR-2 （R=0.37, P=-0.0103）. However, no significant correlation was no- ticed between BMI and VEGF-A or sVEGFR-1 concentrations. Conclusions： A positive correlation determined be- tween the level of antiangiogenic factor and BMI value may be indicative of the linearly growing prevalence of some antiangiogenic factors in patients with metabolic disorders, which may be one of numerous factors contributing to incomplete efficiency of collateral circulation development in patients with PAD.

Design,synthesis and anti-tumor activities of cyclic phosphoramidate mustard-quinazoline conjugates

A series of new cyclic phosphoramidate mustard-quinazoline conjugates were designed and synthesized based on the drug candidate EMB-3, a multi-target-directed ligand against tumor cells, and their anti-tumor activities were evaluated on breast cancer and lung cancer cells. Compound 6d exhibited the best anti-tumor performance with IC5o = 0.6 pM （8-fold of EMB-3） on BT474 breast tumor cells. Compound 6d inhibited epidermal growth factor receptor （EGFIL biomarker for NSCLC） and human epidermal growth factor 2 （HER2, biomarker for breast cancer） with IC50 of 18 nM and 78 nM, respectively. The preliminary pharmacokinetic study revealed that 6d was more stable than EMB-3 during the in vivo metabolism. A single dose per os （PO） administration of 6d in rat model （10 mg/kg） resulted in a moderate tl/2 of 1.7 h. These results indicated that compound 6d was a potential lead compound for the treatment of breast cancer.