基于相位衬度成像的小鼠肺肿瘤三维可视化研究

目的肿瘤的早期发现对患者意义重大。鉴于相位衬度成像对于如肺部这样的软组织成像效果显著,本文提出利用相位衬度成像技术,以微米尺度的空间分辨对肿瘤进行成像,并定量分析,早期发现肿瘤。方法在KM小鼠肺部原位接种人肺癌细胞,培育10d后取出肺组织,利用上海同步辐射光源进行相位衬度成像获取投影图像,拍摄结束后对样本进行切片、HE染色,并光学显微镜观察病理切片。然后利用滤波反投影对获取的投影图进行CT断层重建,并通过病理切片对比验证肿瘤位置,最后通过Amira软件进行三维模型的建立。结果重建出了清晰的三维模型,在模型上可以发现小肿瘤结节的存在,并测量得出该肿瘤体积为0．655mm3。结论相位衬度成像对软组织分辨率高,能发现体积较小的肿瘤．对肿瘤的早期发现有一定意义。     

适用于三维微血管成像的定量分析方法

目的 随着高分辨率三维成像技术的发展,微血管成像已成为临床诊断和实验研究的重要手段.为了在定性观察的基础上定量分析血管形态,本文提出一套完整的图像处理方法,并基于此设计了包括血管分割、血管细化、定量分析在内的血管形态分析流程.方法 首先利用数学形态学方法实现血管结构提取,接着给出计算血管密度、直径等指标的方法,并引入血管的“卷曲度”参数,以衡量血管的扭曲或异常程度.结果 将上述方法应用于1个肝脏血管和3个肿瘤血管灌注成像实例,统计各项形态指标,发现肝脏血管和肿瘤血管在血管密度、分支结构、直径分布以及卷曲度方面均有较大不同.结论 本文方法可有效定量三维血管结构,不仅能够定量较为规则的肝脏血管的形态特征,而且对于尺寸更小、结构更精细的肿瘤血管也能准确地定量分析,因此可以为肿瘤的早期诊断和药物治疗的效果追踪提供重要依据.     

正常及挫伤脊髓血管的三维直视化:同步辐射相衬成像

背景:同步辐射相位衬度成像凭借硬X射线光源的高准直、高相干等特性,其能大幅度提高对软骨细胞及微血管的空间分辨能力。目的:利用高空间分辨率同步辐射相衬成像技术观察大鼠正常及急性损伤脊髓的微血管的形态变化。方法:雄性SD大鼠分为2组:实验组以改良ALLEN’s打击法制作大鼠急性脊髓挫伤模型;伪手术组大鼠只行椎板减压术,不打击脊髓作为正常对照。术后第1天取正常及伤段脊髓标本经甲醛-水杨酸甲酯序贯处理48 h,标本于上海光源BL13W1硬X射线站进行扫描和成像。采集数据以利用VG Studio Max 2.1软件包进行3D图形重建及血管量化分析。结果与结论:相位衬度成像以特有的血管边缘增强效应直观呈现脊髓微血管形态,其结合CT技术(相衬断层成像)从三维视角对脊髓微血管进行直观成像,急性脊髓挫伤后神经组织的破坏伴随着严重的血管结构的损毁,组织损伤及血供的缺失在髓内由中央区向头、尾两侧呈梭性蔓延。三维血管量化数据显示脊髓挫伤后微血管数目及血管灌注容积急剧减少(P0.01)。结果表明无需血管造影的条件下,相衬断层成像可作为一种新的有潜力的超高分辨率可视化技术用于脊髓微血管3D结构成像及量化分析。     

肿瘤新生血管检测的研究进展

肿瘤新生血管是肿瘤营养物质以及代谢产物与机体交换的通路,肿瘤新生血管的生长对肿瘤的生长、恶化、转移、分期等有着重要的影响.以肿瘤血管的概念及其检测为出发点,介绍了肿瘤新生血管的形成、特点和检测意义;着重从肿瘤微血管密度、超声成像、光学成像及核磁共振成像等4个方面综述了肿瘤新生血管检测的研究进展.     

基于X射线相位衬度成像技术的兔眼球血管重建

目的 利用X射线相位衬度成像技术构建兔眼血管网的三维可视化模型,观测兔眼虹膜血管的形态学特征。结果 用硫酸钡对新西兰白兔眼球血管进行造影,利用X射线相位衬度成像技术采集离体兔眼样本的高精度投影图像。图像经由滤波反投影法重建出断层图像,利用Amira 5.2.2软件进行三维重建。方法 高精度投影图像中眼球主干血管清晰连贯,能够观测到部分细小血管的分布及走向,可分辨的最小血管直径约为10 μm;CT扫描图像三维重建后得到兔眼血管网的三维模型,精确到虹膜动脉大环4级分支结构,最小血管直径可达40 μm。结论 利用X射线相位衬度成像技术可以比较清楚地观察到兔眼血管,并且能够在一定程度上构建出血管网的三维可视化模型,为眼球内血管血流动力学分析提供依据,对青光眼的临床研究具有参考价值。     

骨肉瘤肿瘤性粘附因子表达与血管密度及预后的关系

目的：探讨骨肉瘤肿瘤性粘附因子（ＴＡＦ）表达与微血管密度及患者预后关系，方法：应用免疫组化和形态计量方法，检测８０例骨肉瘤ＴＡＦ表达和肿瘤微血管密度，结果：８０例骨肉瘤中３６例ＴＡＦ表达阳性（４０％）阳性反应定位于肿瘤细胞和内皮细胞，以及毛细胞管周围，ＴＡＦ表达与肿瘤微血管密度密切相关（Ｐ〈０．０５），而且ＴＡＦ阳性组生存率显著低于ＴＡＦ阴性组（Ｐ〈０．０５）。结论：ＴＡＦ通过调节血管生成影响骨肉     

Endoglin：肿瘤新生血管的标志物

Endoglin为细胞膜糖蛋白 ,能调节细胞对转化生长因子 β的反应 ,在血管生成的组织和肿瘤组织的血管内皮细胞中肿瘤组织边缘部分血管起源性内皮细胞高度表达。Endoglin的表达与某些肿瘤的预后密切相关 ,可能成为肿瘤的预后指标。应用免疫治疗、基因反义技术阻断肿瘤组织中尚未形成血管的内皮细胞合成Endoglin ,可抑制肿瘤组织微血管的生成 ,阻断肿瘤浸润转移途径 ,将在肿瘤治疗中显示出良好的前景。     

乳腺癌组织内微血管定量与转移的关系

肿瘤内微血管定量是反映乳腺癌预后的重要参数。血管计数和密度级别高的乳腺癌易发生转移或复发。微血管定量与乳腺癌浸润周围血管有关。微血管生成除肿瘤源性因素外，还与肿瘤相关的炎性细胞等因素有关。     

胃癌组织中COX-2表达及其与微血管和微淋巴管密度的关系

目的 研究胃癌组织中COX-2表达的临床病理学意义,同时探讨COX-2与微血管密度(MVD)和微淋巴管密度(MLD)的关系.方法 采用免疫组化EnVision两步法,检测46例胃癌中COX-2的表达,同时标记CD34和D2-40,分别检测胃癌组织中微血管和微淋巴管密度.结果 46例胃癌中COX-2表达的阳性率为80.43%(37/46),COX-2的表达与患者性别、浸润深度无关(P>0.05),而与肿瘤大小、组织学分期、淋巴结转移和临床分期等临床病理参数密切相关(P<0.05);COX-2表达阳性组的平均生存时间明显低于表达阴性组(P<0.05);COX-2表达阳性组的MVD和MLD明显高于COX-2表达阴性组(P<0.05).结论COX-2可能通过促进肿瘤的血管和淋巴管形成而参与了胃癌细胞的浸润转移,导致胃癌患者预后不良.     

原发性星形胶质细胞肿瘤微血管密度和形态与其预后相关性

血管形成是实体性肿瘤生长和增殖的基础，与许多恶性肿瘤的预后直接相关［１］。星形胶质瘤，尤其是高度恶性星形胶质细胞肿瘤具有很强的血管再生能力，其微血管密度与其生物学行为密切相关。我们通过对８７例不同病理类型星形胶质细胞肿瘤中微血管密度和形态的检测，结合...     

Imaging tumor angiogenesis with fluorescent proteins

We have developed three unique mouse models to image angiogenesis with fluorescent proteins, which are described in this review. First, we have adapted the surgical orthotopic implantation (SOI) model to image angiogenesis of human tumors labeled with green fluorescent protein (GFP) transplanted in nude mice. The nonluminous induced capillaries are clearly visible by contrast against the very bright tumor fluorescence examined either intravitally or by whole-body imaging in real time. Intravital images of an SOI model of human pancreatic tumors expressing GFP visualized angiogenic capillaries at both primary and metastatic sites. Whole-body optical imaging showed that blood vessel density increased linearly over a 20-week period in an SOI model of human breast cancer expressing GFP. Opening a reversible skin-flap in the light path markedly reduces signal attenuation, increasing detection sensitivity many-fold and enabling vessels to be externally visualized in GFP-expressing tumors growing on internal organs. The second model utilizes dual-color fluorescence imaging, effected by using red fluorescent protein (RFP)-expressing tumors growing in GFP-expressing transgenic mice that express GFP in all cells. This dual-color model visualizes with great clarity the details of the tumor-stroma interaction, especially tumor-induced angiogenesis. The GFP-expressing tumor vasculature, both nascent and mature, are readily distinguished interacting with the RFP-expressing tumor cells. Using a spectral imaging system based on liquid crystal tunable filters, we were able to separate individual spectral species on a pixel-by-pixel basis. Such techniques non-invasively visualized the presence of host GFP-expressing vessels within an RFP-labeled orthotopic human breast tumor by real-time whole-body imaging. The third model involves a transgenic mouse in which the regulatory elements of the stem cell marker nestin drive GFP. The nestin-GFP mouse expresses GFP in areas of the brain, hair follicle stem cells, and in a network of blood vessels in the skin interconnecting hair follicles. RFP-expressing tumors transplanted to nestin-GFP mice enable specific visualization of nascent vessels in skin-growing tumors such as melanoma. Thus, fluorescent proteins expressed in vivo offer very high resolution and sensitivity for real-time imaging of angiogenesis.     

右旋柠烯乳剂对人胃癌移植瘤生长和转移的抑制作用

目的　研究并探讨右旋柠烯乳剂 (D limonene)对人胃癌生长和转移的抑制作用及其机制。　方法　采用人胃癌BGC 82 3细胞株经反复传代成实体瘤的完整组织块 ,建立人胃癌裸小鼠原位移植瘤模型。将 4 0只荷瘤裸小鼠随机分成 4组 ,移植后第 5d开始分别用生理盐水灌胃、5 氟尿嘧啶 (5 FU)腹腔注射、D limonene灌胃、D limonene灌胃 +5 FU腹腔注射共 7周。第 8周处死动物 ,测量原位肿瘤瘤重并计算抑瘤率 ,检测肿瘤细胞凋亡指数 (AI)、肿瘤微血管密度 (MVD)及免疫组织化学测定肿瘤组织血管内皮生长因子 (VEGF)的表达变化 ,光镜与电镜观察组织细胞超微结构变化 ;观察荷瘤鼠腹膜、肝、其他脏器肿瘤转移及腹水情况。　结果　D limonene组、联用组分别与对照组相比 ,胃癌的原位肿瘤瘤重与抑瘤率、AI、MVD、VEGF下调 ,差异有显著性 (P <0 0 5 )。腹膜、肝转移受到明显抑制 (P <0 0 5 )。　结论　D limonene通过抑制肿瘤微血管形成 ,诱导胃癌细胞凋亡 ,抑制了体内胃癌的生长和转移。     

人肺癌细胞LALU血管生成过程的形态学观察及意义

目的：观察人肺癌细胞血管生成过程的病理形态学、超微结构特点及意义。方法：采用人肺癌细胞LALU皮下移植瘤模型,以病理动态形态学及透射电镜不同时期肿瘤血管生成状态。结果：光镜显示,人肺癌移植瘤第2天至第10天之间可分为血管生成前期和血管形成期,在肿瘤血管生成的第20天出现肺转移灶。电镜显示,人肺癌移植瘤第2天出现成血管细胞,第4－10天,不成熟血管内皮细胞逐渐形成务管腔伴有较完整的新生基底板,内皮细胞趋向成熟发展,第20天肿瘤血管内皮细胞更成熟,部分区域新生毛细血管基底板发育不全或缺陷。在以上全过程中癌细胞突起直接与成血管细胞、血管内皮细胞及血管壁相连。结论：人肺癌细胞可诱导自身肿瘤血管生成,病理形态学及电镜有其特征性形态学改变,且与转移密切相关,为肺癌的血管导向治疗提供了重要依据。     

miR-210对肾透明细胞癌血管生成的影响

目的探讨miR-210对肾透明细胞癌血管生成的影响。方法检测40例肾透明细胞癌组织标本中miR-210的表达,并分析其与相应癌组织中微血管密度(MVD)的关系。通过miR-210反义寡核苷酸(miR-210-ASO)技术抑制肾透明细胞癌细胞系786-O细胞中miR-210表达,RT-q PCR检测转染效果。三维培养实验观察对照组、阴性对照组和miR-210-ASO组肿瘤细胞上清液对人脐静脉内皮细胞(HUVEC)管腔形成数量的影响。建立裸鼠移植瘤模型,应用endomucin免疫荧光染色于激光共聚焦显微镜下观察抑制miR-210对移植瘤生长,血管形成和VEGF表达的影响。结果肾透明细胞癌组织标本中miR-210的表达与微血管密度呈正相关(P0.05),经miR-210-ASO转染的786-O细胞其miR-210表达明显下降(P0.05)。miR-210-ASO组细胞上清液培养的HUVEC细胞管腔形成数量显著少于对照组和阴性对照组(P0.05)。miR-210-ASO组细胞形成的移植瘤体积小于对照组,且微血管数量和VEGF的表达量显著少于对照组(P0.05)。结论抑制miR-210可降低肾透明细胞癌中血管生成数量。     

Imaging cancer dynamics in vivo at the tumor and cellular level with fluorescent proteins

Whole-body imaging with fluorescent proteins has been shown to be a powerful technology to follow the dynamics of metastatic cancer. Whole-body imaging of fluorescent protein-expressing-cancer cells enables the facile determination of efficacy of candidate anti-tumor and anti-metastatic agents in mouse models. GFP-expressing transgenic mice transplanted with the RFP-expressing cancer cells enable the distinction of cancer and host cells and the efficacy of drugs on each type of cell. This is particularly useful for imaging tumor angiogenesis. Cancer-cell trafficking through the cardiovascular and lymphatic systems is the critical means of spread of cancer. The use of fluorescent proteins to differentially label cancer calls in the nucleus and cytoplasm and high-powered imaging technology are used to visualize the nuclear-cytoplasmic dynamics of cancer-cell trafficking in both blood vessels and lymphatic vessels in the live animal. This technology has furthered our understanding of the spread of cancer at the subcellular level in the live mouse. Fluorescent proteins thus enable both macro and micro imaging technology and thereby provide the basis for the new field of in vivo cell biology.     

Comparative evaluation of microvessel density determined by CD34 or CD105 in benign and malignant gastric lesions

Microvessel density (MVD) is regarded as a surrogate marker for angiogenesis and has been used for tumor prognosis. In this study, MVD was identified immunohistochemically by monoclonal antibodies against CD105 and CD34 in the tissues representing gastric carcinoma, chronic gastritis, and hyperplastic polyps, and the results were correlated with clinicopathologic features. The expression of CD105 in the microvessels within benign lesions was barely visible, and MVD was markedly lower than that determined by CD34. CD34 was strongly expressed in the microvessels within hyperplastic polyps and tissues with gastritis. In gastric carcinoma, CD105 expression in microvessels was as high as the MVD, compared with benign lesions. CD105 stained well-formed mature and newly formed immature vessels within the cancer mass. Correlation analysis showed that MVD determined by CD105 correlated with blood vessel invasion, distant metastasis, and formation of ascites. Survival analysis demonstrated an inverse correlation between MVD count and overall survival: patients with MVD counts of 32 or higher survived for a much shorter time than those with counts lower than 32. Multivariate analysis confirmed that MVD determined by CD105 was an independent prognostic factor for survival. Microvessel density determined by CD34 inversely correlated with overall survival, but it did not correlate with other clinicopathologic parameters except formation of ascites. In conclusion, CD34 was universally expressed in blood vessels within benign and malignant tissues, whereas CD105 expression was minimal in benign tissues but stronger in gastric carcinoma. These data suggest that both CD105 and CD34 could be used for quantification of angiogenesis, but preference should be given to CD105 in the evaluation of prognosis in gastric carcinoma.     

肝动脉化疗栓塞联合血管内皮抑素治疗肝癌的试验及临床应用研究

目的了解经肝动脉化疗栓塞术（TACE）联合血管内皮抑素（Endostar）治疗兔VX2肝癌模型和临床原发性肝癌（HCC）患者后,肿瘤微血管密度（MVD）、血管内皮生长因子（VEGF）及CT灌注参数（BF、BV、PS）的变化情况。方法 60例兔VX2肝癌模型均分为三组,单纯TACE治疗组、联合治疗组（TACE联合Endostar）、对照组。40例经病理证实的HCC患者均分为两组,单纯TACE治疗组和联合治疗组（TACE联合Endostar）。分析各组实验动物或病例在治疗前后病理免疫组化（VEGF、MVD）和CT灌注扫描（BF、BV、PS）的改变。结果治疗前,三组兔VX2肝癌模型、两组HCC患者在BF、BV、PS和VEGF、MVD方面差异均无统计学意义。治疗后,联合治疗组（兔VX2肝癌模型、HCC患者）的VEGF、MVD均较单纯TACE组降低,BF、BV、PS均较单纯TACE组升高,差异均存在统计学意义。结论 TACE联合Endostar可以有效抑制兔VX2肝癌模型和临床HCCTACE术后的新生血管的形成,降低肿瘤复发或转移的几率,提高治疗效果。     

中期因子在不同分子分型的乳腺癌中的表达及其临床意义

探讨在5种不同分子分型的乳腺癌中中期因子在组织中的表达,并分析其临床意义。采用免疫组化法检测203例乳腺癌组织的中期因子表达量;采用CD105抗体标记微血管内皮细胞,计算微血管密度(MVD)。结果显示,中期因子与肿瘤的淋巴结转移、临床分期相关性密切。中期因子表达量高的样本具有较高的以CD105表征的微血管密度。与其他4种分子分型相比,中期因子和CD105在基底细胞样型的表达量最高,但是该差异不具有统计学意义。中期因子可以作为乳腺癌预后的标记物应用于临床诊断和治疗,与其他分子亚型的乳腺癌相比,基底细胞样型的乳腺癌可能并不具有独特的血管生成模式。     

Pancreas microenvironment promotes VEGF expression and tumor growth: novel window models for pancreatic tumor angiogenesis and microcirculation

Pancreatic cancer has a poor prognosis, and treatment strategies based on preclinical research have not succeeded in significantly extending patient survival. This failure likely stems from the general lack of information on pancreatic tumor physiology, attributable to the difficulties in developing relevant, orthotopic models that accurately reflect pancreatic cancer in the clinic. To overcome this limitation, we developed abdominal wall windows suitable for intravital microscopy that allowed us to monitor angiogenesis and microvascular function noninvasively during tumor growth in vivo. We used two complementary tumor models in mice: orthotopic (human ductal pancreatic adenocarcinoma, PANC-1, grown in the pancreas), and ectopic (PANC-1 grown in the abdominal wall). We found that orthotopic PANC-1 tumors grew faster than the ectopic tumors and exhibited metastatic spread in the late stage similar to advanced pancreatic cancer in the clinic. Orthotopic PANC-1 tumors expressed vascular endothelial growth factor (VEGF)(121) and VEGF(165), contained higher levels of tumor cell-derived VEGF protein, and maintained vascular density and hyperpermeability during exponential tumor growth. Orthotopic PANC-1 tumors showed lower leukocyte-endothelial interactions in the early stage of growth. In addition, both VEGF(121) and VEGF(165) promoted the growth of PANC-1 cells in vitro. Finally, Anti-VEGF neutralizing antibody inhibited angiogenesis and tumor growth of PANC-1 tumors in both sites. We conclude that the orthotopic pancreas microenvironment enhances VEGF expression, which stimulates growth of PANC-1 tumors (compared with ectopic tumors). The mechanism is autocrine and/or paracrine and also is involved in the maintenance of blood vessels. This comparative system of orthotopic and ectopic pancreatic cancer will provide the rigorous understanding of pancreatic tumor pathophysiology needed for development of novel therapeutic strategies.     

Angiogenesis in the bone marrow of patients with chronic lymphocytic leukaemia

Angiogenesis is a process of formation of new vessels from the preexisting ones. It is involved in many physiological processes, at the same time, however, it is involved also in the progress of tumoral growth. Although a lot is known about angiogenesis in solid tumors where it plays a role in tumoral invasion and its metastatic potential, in hematological malignancies it has been appreciated only recently. However, the results of studies on abnormal angiogenesis in hematological malignancies are inconsistent. Angiogenesis can be studied at different levels; histologically, it is studied in the infiltrated tissues (lymph nodes, bone marrow) and quantified as microvessel density (MVD). The aims of our study were to introduce the method of MVD quantification in the bonemarrow using immunohistochemical detection of endothelial markers (fVIII) and then evaluate MVD in bone marrow samples in a group of patients with chronic lymphocytic leukaemia (CLL) and compare the results with a control group of patients (CON). CLL is a typical malignancy of the hematopoietic tissue but the course and the prognosis of patients with this disease vary considerably. For this reason there is urgent need for novel prognostic markers in order to assess individual patient prognosis and tailor treatment. Angiogenesis is one of the possible markers which may add more informations about the course of this disease. So far only few studies have been published about angiogenesis measured as MVD in CLL patients andthe results are inconsistent. In our study, both the number and the area of microvessels were increased in bone marrow of patients with CLL, but the number and area of sinuses were not. It can be concluded that there are signs of abnormal angiogenesis in bone marrow of patients with CLL but larger study with longer follow-up is needed to give more specific information about prognostic value of these findings.