肿瘤转移关键酶:人乙酰肝素酶氨基端亚基的表达纯化及鉴定

目的:乙酰肝素酶在肿瘤转移的过程中起重要作用,其氨基端小亚基蛋白(Mr为8×103)对其功能活性是不可缺少的。得到此蛋白将有助于深入研究乙酰肝素酶的功能活性。方法:我们从人胎盘cDNA文库中分离乙酰肝素酶蛋白全长编码基因,将乙酰肝素酶氨基端小亚基的基因克隆入pGE-X2TK,转化大肠杆菌进行融合表达。经发酵,纯化,复性得到乙酰肝素酶的氨基端亚基蛋白。结果:得到6.9g纯度>90%的氨基端小亚基融合蛋白。GSTpull-down实验表明,此融合蛋白可与乙酰肝素酶羧基端大亚基蛋白结合。复性后的融合蛋白可被激酶切割为GST蛋白和乙酰肝素酶氨基端亚基蛋白。结论:所得的乙酰肝素酶的氨基端小亚基蛋白为进一步研究该亚基蛋白在细胞内的免疫亚定位及功能奠定了基础。     

乙酰肝素酶、组织蛋白酶D在宫颈癌的表达

目的探讨乙酰肝素酶(Hps)和组织蛋白酶D(CathD)与宫颈癌的发生、发展及预后的关系。方法取我院宫颈癌住院手术患者组织标本89例;宫颈上皮内瘤变(CIN)41例,正常宫颈组织35例。采用免疫组化SP法对乙酰肝素酶和组织蛋白酶D的表达进行检测。结果宫颈癌组织中乙酰肝素酶和组织蛋白酶D的免疫积分明显高于宫颈CIN组织及正常宫颈组织。而在CIN和正常宫颈组织间比较无统计学差异。乙酰肝素酶和组织蛋白酶D的表达与宫颈癌的临床分期、血管浸润和淋巴结转移有关。结论乙酰肝素酶和组织蛋白酶D可能在宫颈癌的发生、发展及预后中起着重要作用。     

重组人乙酰肝素酶多肽片段的表达与纯化

乙酰肝素酶（heparanase，HPA）是一种内源性β-葡萄糖醛酸酯酶，为迄今发现的唯一作用于细胞外基质多聚糖硫酸乙酰肝素蛋白多糖的内切酶。它能够特异性地识别硫酸乙酰肝素（heparan sulfate，HS）结构，并能将其降解为含10—20个糖单位的寡糖链。近20余年，有关乙酰肝素酶的研究表明，乙酰肝素酶对机体的发育、炎症过程、血管生成、肿瘤转移均有重要的作用。目前，人们普遍关注的是恶性肿瘤的发生和发展均伴有乙酰肝素酶的高表达，这意味着：①基底膜的完整性遭到破坏，肿瘤细胞将突破局限转移至他处或穿透基底膜进入血管；②乙酰肝素酶促血管生成作用为侵袭、转移的肿瘤细胞提供了定居、生长所需营养。因此，乙酰肝素酶对肿瘤的发生、发展具有重要作用。     

小肝癌CT动脉期强化特征与乙酰肝素酶等生物学指标关系的研究

目的 探讨小肝癌动脉期强化特征与相关病理学及生物学指标的关系.方法 回顾性分析经手术病理证实的CT动脉期明显强化与无明显强化的小肝癌病例各35例,对照分析其病理学分级、血管生成情况及乙酰肝素酶(HPA)表达情况.结果 小肝癌CT动脉期明显强化与无明显强化2组间微血管密度(MVD)、HPA表达有统计学意义(P<0.05),与病理学分级无统计学意义(P>0.05).结论 小肝癌CT动脉期强化水平与其血管生成情况等生物学行为密切相关,可作为诊断及预后判断的有效指标.     

胰腺癌的CT影像表现与临床病理因素及血管生成的关系

目的研究胰腺癌多层螺旋CT（MSCT）影像表现与临床病理因素及肿瘤血管生成的关系。资料与方法搜集36例经手术病理证实为胰腺导管细胞癌患者的癌肿组织石蜡切片进行特殊免疫组织化学染色。36例患者术前均行MSCT多期动态增强扫描。采用Loyer的CT诊断标准,对受侵胰周血管进行CT分级,并以横断面图像结合多平面重组（MPR）图像,评价肿瘤对周围组织器官的侵犯情况。评价CT像上胰腺癌血管侵犯、胰周侵犯与肿瘤部位、淋巴转移、肝脏转移、UICC分期、病理分级、微血管密度（MVD）、血管内皮生成因子（VEGF）、VEGF—C、金属蛋白酶（MMP）MMP-2、MMP-9的关系。结果对于胰周血管侵犯,统计结果示UICC分期（P＜0．001）,病理分级（P=0．02）,MVD（P＜0．023）,VEGF（P=0．01）,VEGF-C（P=0．016）,MMP-2（P=0．041）有统计学意义。对于胰外侵犯,统计学结果示淋巴转移（P=0．031）,肝脏转移（P=0．025）,UICC分期（P＜0．001）,MVD（P＜0．001）,VEGF（P=0．02）,MMP-2（P=0．01）,MMP-9（P=0．003）有统计学意义。进一步非参数统计显示胰外侵犯,血管侵犯与未侵犯组的平均UICC分期差异均有统计学意义（P＜0．001）,但对于平均病理分级,则差异均无统计学意义（P=0．275,P=1．00）。结论CT影像上胰外侵犯、血管侵犯的表现与肿瘤UICC分期、血管生成关系密切,与肿瘤病理分级无直接联系。血管侵犯和淋巴转移、肝转移并没有明显的相关性,但胰外侵犯和转移密切相关。     

重组人乙酰肝素酶在大肠杆菌中的表达、纯化

目的：表达纯化重组的人乙酰肝素酶（heparanase,HPA）。方法：以表达乙酰肝素酶全长质粒pcDNA3.1-HPA为模板,扩增不包括信号肽的乙酰肝素酶片段,将其插入pET-28a（＋）载体,转化到大肠杆菌BL21（plysS,DE3）中,在起始诱导菌密度、诱导温度、诱导时间、诱导剂浓度4个方面对诱导条件进行了优化,实现了重组人乙酰肝素酶的可溶性表达。采用HisTrapTMcrude亲和层析对目的蛋白进行了初步纯化。结果：得到了初步纯化的HPA蛋白,Western印迹证实表达产物可被乙酰肝素酶抗体和His标签抗体识别,证明表达产物具有乙酰肝素酶的免疫学特性。结论：表达纯化的人HPA蛋白为特异性单抗的制备和鉴定提供了实验材料。     

肝素酶与肿瘤发生发展的研究进展

肝素酶(heparanase,Hpa)是一种糖苷内切酶,可特异性地降解硫酸肝素,破坏细胞外基质和血管基底膜的完整性,释放结合于硫酸肝素上的生长因子、酶类分子等生物活性分子,促进细胞增生和微血管的生长,使肿瘤易于生长转移.国内外的大量研究已证明了肿瘤组织中肝素酶异常高表达,而抑制肝素酶的活性或表达可明显抑制肿瘤的生长、转移和血管发生.     

肿瘤血管生成与抗血管治疗研究进展

化疗、放疗及生物治疗等传统治疗都是针对肿瘤细胞本身。然而,实体瘤不同于血液系统肿瘤,必须通过血管从宿主获取营养。实体瘤由实质和间质组成,后者包括支持组织和血管,显然,针对间质的治疗措施也有间接的抗肿瘤效果。本文结合文献,概述肿瘤血管的特点及抗血管治疗...     

乙酰肝素酶信号肽对其活性功能的影响研究

目的:乙酰肝素酶在肿瘤转移过程中起关键作用,其信号肽在该酶翻译后处理过程中起重要作用.本研究旨在探索乙酰肝素酶在哺乳动物细胞中的表达特性及其信号肽对此酶功能活性的影响.方法:从人胎盘cDNA文库中扩增乙酰肝素酶全长编码基因,克隆入pGEM-T载体中,测序鉴定序列完全正确后,将此基因的全长和不含信号肽基因序列分别克隆入真核表达载体pcDNA4.1/Myc-His中构建pcDNA4.1/Myc-His full HPA和pcDNA4.1/Myc-His part HPA,转化COS-7细胞进行瞬时表达,分析这两种乙酰肝素酶蛋白的表达特性及功能活性.结果:在转染了乙酰肝素酶全基因的COS-7细胞裂解液中检测到该酶的功能活性及大小约53×103(Mr)的目的蛋白免疫印迹表达带,为切割激活后乙酰肝素酶羧基端大亚基与标签蛋白的融合体.在转染了敲除信号肽的乙酰肝素酶基因的COS-7细胞裂解液中测到未被切割激活的大小约65×103(Mr)的目的蛋白免疫印迹表达带,未能检测到该酶的功能活性.结论:在COS-7细胞中表达的完整的乙酰肝素酶蛋白和不含信号肽的乙酰肝素酶蛋白均位于细胞内,没有或很少被分泌到细胞外,提示该酶在正常状态下主要分布在细胞内.含信号肽的酶蛋白在翻译后处理过程中被细胞内的蛋白酶切割成2个亚基而被激活,具有功能活性.而不含信号肽的酶蛋白没有被切割而成为有活性的酶,提示乙酰肝素酶的信号肽对其翻译后加工、激活过程有重要的影响.     

肿瘤血管生成的分子影像学临床应用及研究进展

近年来,人们在基因、分子及细胞层面对抑制肿瘤血管生成的研究取得了长足的进步,并在药物干预和基因治疗方面做了大量有益的尝试,有些动物试验更是取得了相当乐观的结果,与此同时,它的影像学评价方法也日臻完善,作用日益突出.在此,本文将介绍各种成像方法的应用现状,展望各种成像方法在评价抗肿瘤血管生成方面的潜在临床意义.     

AdKDR-CDglyTK融合基因系统对胰腺癌血管生成的抑制作用

目的研究重组腺病毒介导的KDR启动子驱动的CDglyTK融合双自杀基因系统(AdKDR-CDglyTK)对胰腺癌血管生成的抑制作用。方法将20只荷人胰腺癌裸鼠随机分为4组,每组5只。Ⅰ组:注射重组腺病毒AdKDR-CDglyTK与前药5氟胞嘧啶(5-FC)、更昔洛韦(GCV);Ⅱ组:仅注射前药5-FC、GCV;Ⅲ组:仅注射重组腺病毒AdKDR-CDglyTK;Ⅳ组:空白对照,不施加任何处理。采用重组腺病毒AdKDR-CDglyTK瘤体内多点注射,5-FC与GCV腹腔内注射的方法,观察各组治疗效果;微血管密度计数(MVD)分析该系统对胰腺癌血管形成的影响;RT-PCR检测各组的肿瘤组织,以了解有无双自杀基因CDglyTK(目的基因)的表达。结果Ⅰ组裸鼠移植瘤的生长显著受到抑制,Ⅱ、Ⅲ、Ⅳ组肿瘤生长情况无明显差别。MVDⅠ、Ⅱ、Ⅲ、Ⅳ组分别为2.08±0.79,10.01±0.77,9.91±0.63,10.39±1.35,组间差异有统计学意义(F=93.29,P=0.00),Ⅱ、Ⅲ、Ⅳ组与Ⅰ组比较,差异有统计学意义(P<0.05),而Ⅱ、Ⅲ、Ⅳ组之间差异无统计学意义(P>0.05)。RT-PCR结果显示,Ⅰ、Ⅲ组的瘤组织内扩增出2.4kB的目的基因片段,而Ⅱ、Ⅳ组瘤组织内未扩增出目的基因片段。结论AdKDR-CDglyTK联合前药5-FC及GCV在体内可明显抑制裸鼠胰腺癌血管形成和肿瘤生长。     

Vessel imaging with viable tumor analysis for quantification of tumor angiogenesis

Imaging of tumor microvasculature has become an important tool for studying angiogenesis and monitoring antiangiogenic therapies. Ultrasmall paramagnetic iron oxide contrast agents for indirect imaging of vasculature offer a method for quantitative measurements of vascular biomarkers such as vessel size index, blood volume, and vessel density. Here, this technique is validated with direct comparisons to ex vivo micro‐CT angiography and histologic vessel measurements, showing significant correlations between in vivo vascular MRI measurements and ex vivo structural vessel measurements. The sensitivity of the MRI vascular parameters is also demonstrated, in combination with a multispectral analysis technique for segmenting tumor tissue to restrict the analysis to viable tumor tissue and exclude regions of necrosis. It is shown that this viable tumor segmentation increases sensitivity for detection of significant effects on blood volume and vessel density by two antiangiogenic therapeutics (anti‐VEGF and anti‐neuropilin‐1) on an HM7 colorectal tumor model. Anti‐VEGF reduced blood volume by 36 ± 3% (P < 0.0001) and vessel density by 52 ± 3% (P < 0.0001) at 48 h posttreatment; the effects of anti‐neuropilin‐1 were roughly half as strong with a reduction in blood volume of 18 ± 6% (P < 0.05) and a reduction in vessel density of 33 ± 5% (P < 0.05) at 48 h posttreatment. Magn Reson Med 63:1637–1647, 2010. © 2010 Wiley‐Liss, Inc.     

骨髓内皮祖细胞在恶性肿瘤血管生成中的作用

新的血管生成是肿瘤发生和发展的重要病理生理过程。人骨髓中含有内皮祖细胞，骨髓内皮祖细胞通过不同的机制动员进入血液循环，并参与肿瘤的血管生成。深入研究骨髓内皮祖细胞参与肿瘤血管生成的机制将为肿瘤治疗提供新的措施。     

Vessel imaging with viable tumor analysis for quantification of tumor angiogenesis

An “uncorrected” version of the following article was published in the June 2010 issue of MRM. The corrected version of the article is provided here. The publisher regrets the error. Imaging of tumor microvasculature has become an important tool for studying angiogenesis and monitoring antiangiogenic therapies. Ultrasmall paramagnetic iron oxide contrast agents for indirect imaging of vasculature offer a method for quantitative measurements of vascular biomarkers such as vessel size index, blood volume, and vessel density (Q). Here, this technique is validated with direct comparisons to ex vivo micro‐computed tomography angiography and histologic vessel measurements, showing significant correlations between in vivo vascular MRI measurements and ex vivo structural vessel measurements. The sensitivity of the MRI vascular parameters is also demonstrated, in combination with a multispectral analysis technique for segmenting tumor tissue to restrict the analysis to viable tumor tissue and exclude regions of necrosis. It is shown that this viable tumor segmentation increases sensitivity for detection of significant effects on blood volume and Q by two antiangiogenic therapeutics [anti‐vascular endothelial growth factor (anti‐VEGF) and anti‐neuropilin‐1] on an HM7 colorectal tumor model. Anti‐vascular endothelial growth factor reduced blood volume by 36 ± 3% (p < 0.0001) and Q by 52 ± 3% (p < 0.0001) at 48 h post‐treatment; the effects of anti‐neuropilin‐1 were roughly half as strong with a reduction in blood volume of 18 ± 6% (p < 0.05) and a reduction in Q of 33± 5% (p < 0.05) at 48 h post‐treatment. Magn Reson Med 63:1637–1647, 2010. © 2010 Wiley‐Liss, Inc.     

Biological characteristics of gene expression features in pancreatic cancer cells induced by proton and X-ray irradiation

Abstract

Background: Radiation therapy is an important alternative treatment for advanced cancer. The aim of the current study was to disclose distinct alterations of the biological characteristics of gene expression features in pancreatic cancer cells, MIAPaCa-2, following proton and X-ray irradiation.

Materials and methods: Using cDNA microarray, we examined the gene expression alterations of MIAPaCa-2 cells following proton or X-ray irradiation. We also isolated the surviving MIAPaCa-2 cells after irradiation and analyzed their gene expression profiles.

Results: Although the cytocidal effects of both types of irradiation were similar at sufficient doses in vitro and in vivo, the affected gene expression profile alterations of MIAPaCa-2 cells irradiated with protons were distinct from those irradiated with X-ray. Interestingly, clustering analysis of gene expression of the surviving MIAPaCa-2 cells was also completely discernible between the two types of irradiation. However, a similar cytocidal effect was still observed in the proton- and X-ray-irradiated surviving cells after re-irradiation, commonly showing biological effects related to apoptosis and cell cycle processes.

Conclusions: Proton irradiation treatment for pancreatic cancer provides the distinct biological effect of steady gene expression alterations compared to X-ray irradiation; however, surviving cells from both types of irradiation were still susceptible to the cytocidal effects induced by proton re-irradiation treatment.     

Correlation of dynamic contrast enhancement MRI parameters with microvessel density and VEGF for assessment of angiogenesis in breast cancer

PURPOSE: To investigate the association between parameters obtained from dynamic contrast enhanced MRI (DCE-MRI) of breast cancer using different analysis approaches, as well as their correlation with angiogenesis biomarkers (vascular endothelial growth factor and vessel density). MATERIALS AND METHODS: DCE-MRI results were obtained from 105 patients with breast cancer (108 lesions). Three analysis methods were applied: 1) whole tumor analysis, 2) regional hot-spot analysis, and 3) intratumor pixel-by-pixel analysis. Early enhancement intensities and fitted pharmacokinetic parameters were studied. Paraffin blocks of 71 surgically resected specimens were analyzed by immunohistochemical staining to measure microvessel counts (with CD31) and vascular endothelial growth factor (VEGF) expression levels. RESULTS: MRI parameters obtained from the three analysis methods showed significant correlations (P < 0.0001), but a substantial dispersion from the linear regression line was noted (r = 0.72-0.97). The entire region of interest (ROI) vs. pixel population analyses had a significantly higher association compared to the entire ROI vs. hot-spot analyses. Cancer specimens with high VEGF expression had significantly higher CD31 microvessel densities than did specimens with low VEGF levels (P < 0.005). No significant association was found between MRI parameters obtained from the three analysis strategies and IHC based measurements of angiogenesis. CONCLUSION: A consistent analysis strategy was important in the DCE-MRI study. In this series, none of these strategies yielded results for MRI based quantitation of tumor vascularity that were associated with IHC based measurements. Therefore, different analyses could not account for the lack of association.     

MRI of tumor angiogenesis

Angiogenesis has long been established as a key element in the pathophysiology of tumor growth and metastasis. Increasingly, new molecularly targeted antiangiogenic drugs are being developed in the fight against cancer. These drugs bring with them a need for an accurate means of diagnosing tumor angiogenesis and monitoring response to treatment. Imaging techniques can offer this in a noninvasive way, while also providing functional information about the tumor. Among the many clinical imaging techniques available, MRI alone can provide relatively good spatial resolution and specificity, without ionizing radiation and with limited side effects. Arterial spin labeling (ASL) and blood oxygenation level-dependent (BOLD) imaging techniques can be employed to confer indirect measures of angiogenesis, such as blood flow and blood volume, without the need for external contrast agents. Dynamic contrast-enhanced (DCE)-MRI is rapidly emerging as a standard method for directly measuring angiogenesis during angiogenesis-inhibitor drug trials. As macromolecular MR contrast agents become available, they will inevitably be utilized in the assessment of tumor perfusion and vessel permeability. Meanwhile, technological advances have made imaging at a molecular level a possibility. They have brought the potential to directly target MR contrast agents to markers of angiogenesis, such as the alpha(v)beta(3) integrin. Before this is used clinically, however, substantial gains in sensitivity brought about by improved coils, pulse sequences, and contrast agents will be needed. Herein we discuss the techniques currently available for MRI of angiogenesis, along with their respective advantages and disadvantages, and what the future holds for this evolving field of imaging.     

Pancreatic cancer: correlation of MR findings, clinical features, and tumor grade

PURPOSE: To assess the frequency of occurrence of poorly-marginated and focally-defined pancreatic ductal adenocarcinoma by MRI and to determine whether these appearances correlate with clinical features and histopathological grade. MATERIALS AND METHODS: Institutional review board with waiver of informed consent was obtained for this HIPAA compliant study. A total of 33 patients (16 female, 17 male, mean age = 63.5 +/- 12.8, ranging from 41 to 80 years) with histopathologically-proven pancreatic ductal adenocarcinoma who underwent MR examination between August 2000 and February 2005 were retrospectively evaluated. Clinical data and histopathological tumoral grade were obtained from clinical charts; nine of 33 patients were excluded of the histopathological evaluation since their diagnosis was performed by fine needle aspiration biopsy and it was not possible to obtain the histopathological grade. Two radiologists reviewed all cases independently to identify whether cancers were poorly-marginated or focally-defined. Agreement between radiologists was assessed using the kappa coefficient. The overall correlation between imaging findings, clinical features, and histopathological grade was assessed with contingency tables using the Fisher's exact test. RESULTS: Of the 33 patients, nine (27.2%) were classified as poorly-marginated and 24 (72.8%) as focally-defined. Agreement between the two reviewers was excellent (k = 0.92, 95% confidence interval (CI) = 0.78-1.0). Poorly-marginated cancers exhibited well- to moderately-differentiated histopathology in 71.4% of cases, while focally-defined cancers had well- to moderately-differentiated histopathology in 17.6% of cases, P = 0.02. CONCLUSION: A poorly-marginated appearance of pancreatic ductal carcinoma on MRI is not uncommon. These cancers exhibited statistically significant moderate- to well-differentiated histopathology compared to focally-defined cancers.     

早期胃癌内镜、病理检查特征及其浸润深度相关性研究

 目的 回顾性分析早期胃癌(early gastric cancer,EGC)内镜、病理检查特征及其与浸润深度的关系。方法 选择经胃镜下诊断+活组织病理检查及手术后病理确诊为EGC的72例患者资料,分析内镜诊断及其临床病理特征,探讨EGC的内镜、临床病理特征及其与浸润深度相关性。结果 EGC患者的性别、年龄、主要症状、分化程度、内镜下形态学分型均与浸润深度无相关性。病灶大小直径≥2 cm者,15例侵及黏膜下层,占60.0%,病灶大小与浸润深度存在统计学关联(χ²＝22.273,P=0.000, Cramer’s V=0.556);病灶部位与浸润深度存在统计学关联(χ²＝17.743,P=0.000,Cramer’s V =0.496),上部侵及黏膜下层占77.78%,而中部及下部仅12例侵及黏膜下层(19.05%)。结论 EGC的病灶部位、病变大小与肿瘤浸润深度密切相关,探索EGC浸润深度相关的内镜及病理表现规律,可为EGC的检出、治疗与预后判断提供进一步指导依据。