肝素酶介导的肝癌细胞与血管内皮细胞黏附及穿内皮迁移

目的:探讨肝素酶(heparanase,HPSE)对肝细胞癌(hepatocellular carcinoma,HCc)细胞与血管内皮细胞黏附及穿内皮迁移的影响.方法:选用人正常脐静脉内皮细胞株HUVEC-C,肝LO-2细胞,肝癌HepG2、BEL-7402和HCCLM3细胞,用Real-time PCR筛选高表达HPSE的HCC细胞;设计4个HPSE基因RNA干扰(RNA interference,RNAi)序列、构建质粒,并筛选出最优RNAi质粒;用该RNAi质粒转染高表达HPSE的HCC细胞,同时设空白对照组、阴性对照组和未转染的HCC细胞组;黏附试验观察各组HCC细胞与HUVEC-C细胞的黏附情况;Transwell小室法观察HCC细胞穿HUVEC-C细胞迁移情况;癌细胞均以0.25％玫瑰红染色、光镜下观察;酶标仪法分别测定脱色后各组细胞D值并分析比较.结果:HCCLM3细胞HPSEmRNA表达水平显著高于肝LO-2、HepG-2和Bel-7402细胞[(5.72±0.62) vs (1.05 ±0.09)、(2.65±0.31)和(3.43±0.58),均P＜0.01)l;设计的4个HPSE之RNAi载体,以siHPSE-3158对HPSE基因表达的抑制效果最佳(P＜0.05).RNAi组HCCLM3细胞与HUVEC-C细胞黏附率显著低于空白对照组、阴性对照组和未转染的HCCLM3细胞组[(0.31 ±0.04) vs (0.46±0.06)、(0.45 ±0.05)和(0.64±0.09),均P＜0.01)].RNAi组HCCLM3细胞穿HUVEC-C细胞迁移率也显著低于空白对照组、阴性对照组和未转染的HCCLM3细胞组[(0.28 ±0.03) vs (0.41 ±0.04)、(0.41±0.05)和(0.43±0.05),均P＜o.05)].结论:HPSE参与介导HCC细胞与血管内皮细胞的黏附及HCC细胞穿内皮迁移,可能是HCC微血管捕获、血行转移的重要因素.     

肝癌新生血管的形态学异常及其临床意义

 目的 研究肝细胞癌（HCC）新生血管的形态学异常。方法 采用免疫组化SABC法检测56例HCC组织及其相应癌旁肝组织和6例正常肝组织中CD34和Endoglin的表达,并采用透射电镜观察微血管超微结构的改变。结果 56例HCC组织中CD34和Endoglin表示的MVD值分别是127.1±5.9和64.6±11.1,而癌旁肝组织的MVD值分别是7.1±1.3和6.1±1.2。HCC组织的MVD值高于相应的癌旁肝组织,差异具有显著意义（P〈0.01）。HCC组织中微血管结构松散,通透性明显增加,可见微血管内微癌栓形成。结论 HCC组织中新生血管生成明显增加,而且这些新生的微血管在结构上是有缺陷的,这为癌细胞的转移提供了门户。     

肝癌诱导特异性血管新生实验方案的建立

目的 建立一套用于研究和评价抗肝癌血管新生的实验方案。方法 以人脐静脉内皮细胞（HUVEC）为细胞模型,以正常胎肝细胞系L02为对照,应用荧光定量的Boyden小室、划痕实验检测肝癌条件培养基（HCM）、肝癌细胞系HepG2等诱导作用下对HUVEC迁移的影响;应用荧光定量的粘附实验检测上述诱导作用对HUVEC的粘附作用;应用CFSE／PKH-26 染色流式细胞术检测上述诱导作用对HUVEC增殖的作用;应用小管形成实验、Matrigel 栓实验检测上述诱导作用对HUVEC小管形成行为及对SCID小鼠体内血管新生的影响。结果 体外实验显示,HCM能够比正常条件培养基（NCM）更能诱导HUVEC的趋化和迁移运动,差异有统计学意义（P＜0.05）; HepG2较L02更容易粘附HUVEC细胞,差异有统计学意义（P＜0.05）; HCM较NCM更能促进HUVEC的增殖,与L02相比,HepG2和HUVEC共培养更能促进人脐静脉内皮的增殖,差异有统计学意义（P＜0.05）; HCM和/或HepG2与HUVEC 共培养均有利于HUVEC形成管型,而NCM和/或L02与HUVEC共培养对HUVEC形成管型无明显影响。Matrigel栓实验显示,HepG2较L02更能促进SCID小鼠体内的血管新生。结论 以上一系列改良的体内、外经典实验能够用于抗肝癌血管新生药物的研究和评价。     

肝细胞癌化疗栓塞后血清血管内皮细胞生长因子与转移关系的研究

目的　前瞻性评估肝细胞癌 (HCC)经导管化疗栓塞 (TACE)前后血清血管内皮细胞生长因子 (VEGF)表达变化与复发转移的关系。方法　采用酶联免疫夹心法 (ELISA)对 3 0例HCC患者分别于术前、术后 3d和 4周测量血清VEGF水平 ,TACE术后 3个月评估复发转移发生情况。结果　 3 0例HCC患者术前血清VEGF表达范围为 154.47± 90 .17pg/ml ,术后血清VEGF表达较术前增高 (P <0 .0 5)。在碘油分布不均匀及门静脉癌栓组中 ,血清VEGF表达增高。追踪半年后 ,血清VEGF水平升高者中有 74%复发 ,而血清VEGF表达下降者无一例复发。结论　HCC患者TACE后血清VEGF表达增加 ,与复发转移发生有关     

原发性肝癌微血管密度的研究

目的探讨原发性肝癌中微血管密度(MVD)与肿瘤生物学行为之间的关系.方法应用免疫组织化学SABC技术,对51例肝癌组织中FⅧRAg进行免疫特异性染色,并计数肿瘤组织内微血管密度(MVD).结果①肝癌＞5cm、无包膜、有肝内转移、Child分级为B级和C级的MVD明显高于肝癌≤5cm、有包膜、无肝内转移、Child分级为A级(P＜0.05～0.01);②有门脉侵犯及淋巴结转移肝癌的MVD虽高于无门脉侵犯及淋巴结转移肝癌,但差异无显著性(P＞0.05);③MVD与病理分级、腹水和AFP无关(P＞0.05).结论MVD在肝癌的生长和转移中起到重要的作用,可作为肝癌预后的一个有用的预测指标.     

血管生长因子与肝细胞性肝癌的关系

血管生长因子对于肿瘤的血管新生具有重要的作用，因此也影响肿瘤的发生、发展、浸润和转移等生物学行为。肝细胞癌(hepatocellular carcinoma，HCC)是富血供的肿瘤，血管生长因子对其微血管的生成影响较大。介绍了与HCC有关的血管生长因子，并阐述它们的作用机制，以及它们对于HCC生物学行为的影响。     

血管生成抑制剂TNP-470抑制肝癌生长和转移的实验研究

目的　研究血管生成抑制剂TNP 470对肝细胞癌生长和转移的抑制作用。方法　把高转移人肝癌模型(LCI D2 0 )的肿瘤组织小块种植于裸鼠皮下 ,将 2 4只裸鼠随机分成对照组、治疗组。第 2天起分别给予溶剂 ( 3%酒精 )、TNP 470 ( 30mg kg)隔天皮下注射 ,共 8次。 结果　对照组、治疗组皮下瘤重分别为 ( 2 0 4± 0 34)g、( 0 98± 0 34) g(P<0 0 0 1) ;两组AFP分别为 ( 76 8 6± 2 82 3) μg L ,( 93 4±5 8 6 ) μg L (P <0 0 0 1) ,两组肺转移率分为5 0 % ( 6 12 )、8 3 % ( 1 12 ) (P <0 0 5 )。结论　血管生成抑制剂TNP 470能显著抑制肝细胞癌的生长和转移     

miR-375调控PDGFC基因表达抑制肝癌血管生成

  目的  miR-375已被证明能够抑制肝癌细胞增殖、迁移和侵袭,促进肝癌细胞凋亡,本研究旨在探索miR-375对肝癌新生血管生成的影响及其分子机制。  方法  利用血管生成相关的功能实验探索miR-375对肝癌新生血管生成的影响;使用生物信息学方法预测miR-375潜在的与血管生成相关的功能靶基因;在肝癌细胞系中过表达和下调表达miR-375,验证miR-375对靶基因的调控作用,并利用双荧光素酶报告实验明确其分子机制;靶基因功能挽救实验明确miR-375通过调控靶基因的表达发挥抑制肝癌新生血管生成的作用。  结果  miR-375能够抑制肝癌新生血管生成;血小板源性生长因子C（PDGFC）是miR-375的潜在功能靶基因;miR-375能够直接作用于PDGFC基因mRNA 3'-非编码端（3'-UTR）而抑制PDGFC基因表达;miR-375能够通过抑制PDGFC基因表达抑制肝癌新生血管生成。  结论  miR-375能够调控PDGFC基因表达抑制肝癌新生血管生成。      

血管内皮生长因子在肝癌血管内皮细胞体外管腔形成中量效和时效局限性的实验研究

目的：利用体外管腔形成模型,研究血管内皮生长因子（VEGF）对肿瘤血管内皮细胞（TECs）调控的精准性,探讨以 VEGF 为靶点的抗肿瘤药物在临床应用中出现缺陷的潜在机制。方法采用 CD31免疫磁珠分选人肝癌标本中的 TECs,在含 Matrigel 基质胶的96孔板中,分别与不同浓度 VEGF 共培养,观察不同时间的 TECs 体外管腔形成能力,以正常人脐静脉血管内皮细胞（ HUVECs）为对照。结果1）体外分离培养的 TECs 拟血管内皮细胞梭状形态,其96％的细胞表达内皮细胞特异性标记 CD31;2）TECs 在微小量（基础培养条件,2 ng·mL -1）VEGF165时,体外管腔形成较少或不成腔,而对照组 HUVECs 却能形成明显的管腔并分支成网;当附加10 ng·mL -1 VEGF165时,在4 h 观察到 TECs 如同 HUVECs 形成了管腔,在6 h 出现明显的分支网;然而,当附加20 ng·mL -1 VEGF165时,在6 h 观察到 TECs 形成的管腔明显减少;统计分析显示,10 ng·mL -1 VEGF165组 TECs 成管能力比2 ng·mL -1 VEGF165组高出6倍（ P ﹤0.001）,而20 ng·mL -1 VEGF165组 TECs 成管能力显著下降至10 ng·mL -1 VEGF165组的4.5倍（P ﹤0.001）;3）10 ng·mL -1 VEGF165组在培养4、6、8 h 均可见 TECs 管腔形成,但以6 h 为显著;在20 h 时,TECs 的管腔消失,而对照组 HUVECs 却还有明显的管腔分支网。结论肝癌 TECs 体外管腔形成对 VEGF165既有依赖性但又有量-效局限性,过高 VEGF 浓度能抑制性影响 TECs（而不是 HUVECs）管腔形成能力。在 VEGF 的刺激下,TECs 体外管腔形成的时效性短于 HUVECs。我们的实验结果提示,临床应用抗 VEGF 的抗肿瘤药物疗效不一可能与 VEGF 在 TECs 体外管腔形成的量效和时效的局限性有关。     

循环内皮细胞与肿瘤血管生成

循环内皮细胞（circulating endothelial cells,CECs）是指外周血中测得的血管内皮细胞（vascular endothelial cells,VEC）。20世纪70年代,Bou—vier和Hladovec在动物试验中发现了这种细胞,并提出了CECs的概念。CECs在正常人外周血中数量极少,而在动脉粥样硬化、糖尿病、红斑狼疮等疾病中明显增加。多项研究表明,CECs数量的增加与内皮细胞受损明显相关,因此被认为是判断血管内皮细胞损伤情况的最特异而直接的指标。     

Chemotherapy and the tumor microenvironment: the contribution of circulating endothelial cells

Anti-angiogenic drugs, alone or in combination with chemotherapeutics, are increasingly used by medical oncologists. In many cases, however, their mechanism of action and the tailoring of optimal dosage/schedule are still elusive. Circulating endothelial cell (CEC) and progenitor (CEP) number and viability are modulated in a large series of diseases including cancer, and look promising as surrogate biomarkers for the definition of the optimal biological dose of anti-angiogenic drugs and for patients’ stratification. Along with CECs and CEPs, potential EC- and CEP-related surrogate molecular markers such as VE-Cadherin and CD133 are currently under preclinical and clinical investigation.     

Circulating endothelial progenitor cells (EPC) for tumor vasculogenesis in gastric cancer patients

It has been suggested that vasculogenesis by endothelial progenitor cells (EPC) as well as angiogenesis play an important role in the production of blood vessels in neoplasm. The present study was designed to isolate and characterize the EPC in gastric cancer patients as a tumor specific angiogenesis marker. The cells derived from CD34 positive PBMC presented with a cobblestone appearance at 28 days, revealing differentiation into endothelial cells. They were also positive to the LDL-uptake reaction, showing that they have biological endothelial cell functions. These cells demonstrated tube formation, showing their ability to participate in neovascularization. The cells derived from CD34 positive PBMC expressed CD133 and demonstrated telomerase activity, showing the stem cell character. In xenograft model, EPC derived from CD34 positive PBMC mobilized mainly into tumor area after being injected through tail vein. With isolation, ex vivo amplification and characterization of EPC from gastric cancer patients receiving chemotherapy, endothelial progenitor cells may be used as a candidate prognostic and predictive biomarker for cancer.     

Phenotypic characteristics of cells derived from precursors of human melanoma

Of 66 specimens from benign melanocytic nevi, including common acquired and congenital nevi, Spitz tumors (epithelioid cell nevi), and melanocytic nevi with dysplasia, 57 could be grown in tissue culture. The cultured cells were identified as melanocytes by the presence of premelanosomes and melanosomes. Cells from 28 of 32 nevus cultures grew in an anchorage-independent way in soft agar with a colony-forming efficiency between 0.001 and 1%. Clones derived from single cells and soft agar-selected colonies showed marked phenotypic heterogeneity, but all had a limited life span and did not undergo transformation in culture. These cells were nontumorigenic in nude mice. Cultured nevus cells expressed antigens present on melanoma but absent on normal fibroblasts and/or melanocytes as tested with monoclonal anti-melanoma antibodies. The anti-melanoma antibodies bound equally well to dysplastic, congenital, and common acquired nevi. Antigens are released by nevus cells similar to melanoma cells.     

Biologic markers of angiogenesis: circulating endothelial cells in patients with advanced malignancies treated on phase I protocol with metronomic chemotherapy and celecoxib

Preclinical studies demonstrate anti-angiogenic activity of low doses of chemotherapy; selective cox-2 inhibitors are also inhibitors of angiogenesis. Animal data indicates the presence of circulating endothelial cells (CEC), tumor-derived activated endothelial cells (AEC) and endothelial cell progenitors (ECP). Bone marrow-derived ECP have been shown to be incorporated into tumor vasculature. We conducted two combination Phase I studies of celecoxib with either cyclophosphamide or etoposide. Exploratory correlative studies were performed to evaluate the detectability of CEC, AEC and ECP in patients treated with these anti-angiogenic combinations. Patients were treated with oral cyclophosphamide at 50 mg daily or etoposide at 50 mg daily. Celecoxib was given at 400 mg twice daily. Blood samples were collected on days 0, 7, 28 and monthly until disease progression. Blood from healthy volunteers was collected on days 0 and 28. Peripheral mononuclear cells (PMNC) were isolated and stained with fluorescent antibodies and analyzed utilizing 5-color flow cytometry. Forty-four heavily pretreated patients (20 F; 24 M) with various solid tumors were enrolled. Median age was 65 (23-72). Therapy was well tolerated. No responses were seen. Six patients had stable disease for at least 16 weeks. The longest duration on therapy is 420 days in a patient with metastatic thymoma. Pre-therapy CEC were detected in cancer patients and normal controls with mean concentrations of 0.47 cells/uL and 0.14 cells/uL, respectively. Mean ECP in patients and controls were 0.09 cells/ uL and 0.03 cells/uL, respectively. No AEC were detected. No consistent changes were seen in CEC or ECP during therapy. The combinations of oral cyclophosphamide or etoposide at 50 mg daily with celecoxib at 400 mg twice daily are well tolerated with occasional prolonged disease stabilizations observed. CEC and ECP are detectable in cancer patients but their levels did not change significantly during therapy with our regimen. Further evaluation of CEC and ECP in patients treated with clinically more active anti-angiogenic therapies would be of interest.     

Pilot study on the interaction between B16 melanoma cell-line and bone-marrow derived mesenchymal stem cells

Bone-marrow derived mesenchymal stem cells (BMSCs) have the potential to differentiate into osteocytes, chondrocytes, adipocytes and endothelial cells. The interaction between BMSCs and epithelial tumor cell was enhanced on proliferation. Our previous study had shown that BMSCs maybe participate in angiogenesis in melanoma in vivo. The aim of this study was to investigate the interaction between B16 melanoma cells and BMSCs in vitro, the mechanism of BMSCs participating in melanoma angiogenesis in vivo is unclear, so a co-culture system containing BMSCs and B16 melanoma cells, based on transwell indirect model, was established, and the interaction between BMSCs and B16 melanoma cells was studied in vitro. In our study, BMSCs were generated out of bone marrow from C57 mouse, isolated BMSCs were positive for the markers CD105, CD90, CD73, CD44 and CD166 and negative for endothelial markers, which acquired endothelial phenotype (including the expression of VEGFR-1, VEGFR-2, Factor VIII) after co-culture with B16 melanoma cells; at the same time, B16 melanoma cells also up-regulated the expression of VEGF-a, VEGFR-1, VEGFR-2 and Factor VIII. The proliferation rate of B16 melanoma cells and BMSCs were also found to be increased. We could show the differentiation of BMSCs into cells with phenotypic features of endothelial cells. BMSCs promoted proliferation of tumor cells and improved the microenvironment in tumor. Our study suggests that the BMSCs may play an important role in tumor angiogenesis.     

Norcantharidin anti-angiogenesis activity possibly through an endothelial cell pathway in human colorectal cancer

The present study was based on the unexpected discovery that norcantharidin exerted anti-angiogenesis activity when effects on growth of human colon cancer were studied. The aim was to further verify this finding and explore possible mechanisms using a tumor xenograft model in nude mice. We confirmed that norcantharidin (5 or 15 mg/kg) could inhibit angiogenesis of human colon cancer in vivo. In vitro, crossing river assay, cell adhesion assay and tube formation assay indicated that NCTD could reduce the migration, adhesion and vascular network tube formation ability of HUVECs. At the same time, the expression levels of VEGF and VEGFR-2 proteins which play important roles in angiogenesis were reduced as examined by western blotting analysis. Taken together, the results firstly showed NCTD could inhibit angiogenesis of human colon cancer in vivo, probably associated with effects on migration, adhesion and vascular network tube formation of HUVECs and expression levels of VEGF and VEGFR-2 proteins.     

Far-infrared radiation inhibits proliferation,migration, and angiogenesis of human umbilical vein endothelial cells by suppressing secretory clusterin levels

Far-infrared (FIR) radiation is known to lessen the risk of angiogenesis-related diseases including cancer. Because deficiency of secretory clusterin (sCLU) has been reported to inhibit angiogenesis of endothelial cells (EC), we investigated using human umbilical vein EC (HUVEC) whether sCLU mediates the inhibitory effects of FIR radiation. Although FIR radiation ranging 3–25 μm wavelength at room temperature for 60 min did not alter EC viability, further incubation in the culture incubator (at 37 °C under 5% CO₂) after radiation significantly inhibited EC proliferation, in vitro migration, and tube formation in a time-dependent manner. Under these conditions, we found decreased sCLU mRNA and protein expression in HUVEC and decreased sCLU protein secreted in culture medium. Expectedly, the replacement of control culture medium with the FIR-irradiated conditioned medium significantly decreased wound closure and tube formation of HUVEC, and vice versa. Furthermore, neutralization of sCLU with anti-sCLU antibody also mimicked all observed inhibitory effects of FIR radiation. Moreover, treatment with recombinant human sCLU protein completely reversed the inhibitory effects of FIR radiation on EC migration and angiogenesis. Lastly, vascular endothelial growth factor also increased sCLU secretion in the culture medium, and wound closure and tube formation of HUVEC, which were significantly reduced by FIR radiation. Our results demonstrate a novel mechanism by which FIR radiation inhibits the proliferation, migration, and angiogenesis of HUVEC, via decreasing sCLU.     

CD133+ liver cancer stem cells modulate radioresistance in human hepatocellular carcinoma

CD133 is a cancer stem-cell (CSC) marker associated with radioresistance and chemoresistance in various cancers. In the present study, CD133-expressing liver cancer cells following radiation exposure showed higher activation of MAPK/PI3K signaling pathway and reduction in reactive oxygen species levels compared to CD133⁻ cells. The in vivo study with a xenograft model showed increased tumor formation in irradiated CD133⁺ cell-injected nude mice compared to the CD133⁻ group, suggesting that CD133 contributes to radioresistance in HCC. Therefore, CD133-expressing liver cancer cells have anti-apoptotic and radioresistance properties that may be useful to improve anti-cancer treatments, including chemotherapy/radiotherapy of HCC.