口虾蛄提取物对人鼻咽癌细胞P53、COX-2和VEGF表达的影响

目的: 研究口虾蛄提取物(EOS)对人低分化鼻咽癌细胞系(CNE-2Z)中P53、环氧化酶2(COX-2)和血管内皮生长因子(VEGF)表达的影响及意义。方法: 不同浓度EOS(0 mg/L、125 mg/L、250 mg/L和500 mg/L)作用CNE-2Z细胞24 h后,Western blotting检测P53蛋白表达,免疫细胞化学法及RT-PCR法分别检测COX-2 、VEGF蛋白和mRNA的表达。结果: CNE-2Z细胞中P53蛋白、COX-2和VEGF蛋白和mRNA表达均随EOS作用浓度增高而逐渐降低,呈量效关系(P<0.01);且COX-2 和VEGF表达量呈正相关(P<0.05),COX-2 和P53表达量亦呈正相关(P<0.05)。结论: EOS可能通过抑制CNE-2Z细胞中P53蛋白的表达,干扰COX-2和VEGF的表达而发挥其抗肿瘤作用的。     

XAV939抑制人肝癌HepG2细胞血管生成拟态的形成

目的初步研究XAV939对人肝癌Hep G2细胞血管生成拟态形成的影响及其可能的机制。方法实验分为对照组和实验组(0.5和1μmol/L XAV939分别处理Hep G2细胞48 h);体外成管实验检测各组细胞形成血管拟态的能力,RT-PCR检测ZEB1及MMP-7 mRNA的表达,Western blot检测β-catenin、ZEB1和MMP-7蛋白的表达。结果对照组与实验组形成管状结构数目分别为9.67±0.70,5.67±0.64(0.5μmol/L)和2.27±0.81(1μmol/L),体外形成管道结构的数目减少(P0.01);实验组ZEB1及MMP-7 mRNA表达和ZEB1、MMP-7及β-catenin蛋白表达均较对照组减少(P0.05)。结论 XAV939能有效抑制人肝癌Hep G2细胞血管生成拟态的形成。     

β-榄香烯抑制B16恶性黑色素瘤细胞的EPHA2表达和血管生成拟态

目的研究β-E对B16恶性黑色素瘤细胞上皮细胞激酶(epithelial cell kinase,EphA2)表达和血管生成拟态的影响及其相关机制。方法培养B16细胞,建立体外恶性黑色素瘤模型,应用qRT-PCR法和Western-blot法检测β-E对B16细胞EPHA2 mRNA和蛋白表达影响,应用Matrigel诱导血管生成实验检测β-E对B16细胞血管生成拟态生成能力的影响。结果成功建立体外恶性黑色素瘤模型,β-E显著抑制B16细胞EPHA2mRNA和蛋白表达,抑制B16细胞血管生成拟态生成能力。结论β-E可在治疗恶性黑色素瘤中起重要作用。     

VEGF通过上调CCN2促HUVEC迁移和血管生成

目的 探讨血管内皮生长因子(VEGF)诱导的成骨细胞中结缔组织生长因子(CTGF/CCN2)对人脐静脉血管内皮细胞(HUVECs)的影响.方法 用Real time PCR法及ELISA法检测VEGF诱导成骨细胞(OSE)中CCN2含量;制备成骨细胞(OSE)上清液;将细胞分为control组、OSE组和VEGF-OSE组(n=3).用小干扰RNA (siRNA)转染法抑制成骨细胞中CCN2的表达;Transwell法检测内皮细胞迁移;Matrigel实验检测管样结构形成能力.结果 VEGF呈时间和剂量依赖性上调成骨细胞中CCN2 mRNA和蛋白的表达;CCN2可促进内皮细胞的迁移和管样结构形成(P＜0.05),当CCN2被siRNA基因沉默或者加入CCN2抗体后,CCN2对内皮细胞迁移和管样结构形成的促进作用均受到明显抑制(P＜0.05).结论 VEGF通过上调成骨细胞中CCN2的表达,促内皮细胞(HUVECs)的迁移和血管生成.     

转录因子Bach1对人微血管内皮细胞的作用研究

 目的:研究转录因子Bach1对人微血管内皮细胞功能的影响。方法: 利用小干扰RNA（small interfering RNA,siRNA）细胞转染技术下调内皮细胞Bach1表达;用Matrigel管腔形成实验检测内皮细胞体外血管新生的能力;用Transwell小室法检测细胞迁移;用CCK-8法测定细胞增殖;用实时荧光定量PCR、Western blotting和ELISA法检测细胞中血红素氧合酶1（heme oxygenase 1,HO-1）和血管内皮细胞生长因子（vascular endothelial growth factor, VEGF）mRNA 和蛋白的表达情况;用转染报告基因的方法检测VEGF基因的转录活性。结果: 下调内皮细胞Bach1表达明显促进人微血管内皮细胞迁移和管腔形成能力,对内皮细胞增殖能力无明显影响;抑制Bach1表达促进内皮细胞HO-1 mRNA 和蛋白的表达,增加VEGF 转录活性及mRNA和蛋白的表达。结论: 抑制转录因子Bach1表达可增加内皮细胞HO-1和VEGF的表达,促进人微血管内皮细胞迁移和管腔形成,提示Bach1是负性调控血管新生的因子。     

口虾蛄提取物对人鼻咽癌细胞端粒酶活性的影响

目的:研究口虾蛄提取物(EOS)对人低分化鼻咽癌细胞系(CNE-2Z)端粒酶活性的抑制作用及其可能机制。方法:MTT法检测不同浓度的EOS对CNE-2Z细胞增殖能力的影响;端粒酶重复序列扩增-酶联免疫吸附法(TRAP-ELISA)检测各组细胞端粒酶活性,RT-PCR法检测端粒酶逆转录酶(hTERT)mRNA表达,Western blotting检测c-Myc蛋白表达。结果:EOS对CNE-2Z细胞增殖具有抑制作用,且呈剂量依赖性(P0.01);EOS处理组CNE-2Z细胞端粒酶的活性、hTERT mRNA和c-Myc蛋白的表达水平均低于对照组(P0.01),也呈剂量依赖性,并且hTERT mRNA的下调与c-Myc的抑制存在相关性(P0.05)。结论:EOS可能通过下调细胞c-Myc表达而抑制了hTERT mRNA的转录,以致端粒酶的活性降低而抑制CNE-2Z细胞增殖。     

二氢杨梅素对绒毛膜癌细胞增殖和迁移能力的影响

目的 探讨二氢杨梅素（DMY）对绒毛膜癌（绒癌）JEG-3及JAR细胞增殖和迁移能力的影响。 方法 MTT法检测不同浓度的二氢杨梅素(0 mg/L, 20 mg/L, 40 mg/L, 60 mg/L, 80 mg/L)作用一定时间后,对绒癌JEG-3和JAR细胞增殖能力的影响;细胞划痕实验和Transwell法检测不同浓度二氢杨梅素(0 mg/L, 40 mg/L, 60 mg/L, 80 mg/L)分别作用绒癌JEG-3细胞及JAR细胞一定时间后,对其迁移能力的影响;Real-time PCR和Western blotting方法分别检测不同浓度二氢杨梅素(0 mg/L, 40 mg/L, 60 mg/L, 80 mg/L)作用绒癌JEG-3及JAR细胞后,基质金属蛋白酶2（MMP-2）mRNA和蛋白表达水平的影响。 结果 不同浓度二氢杨梅素作用绒癌JEG-3和JAR细胞24 h和48 h后,随着二氢杨梅素浓度增加,对JEG-3和JAR细胞增殖抑制作用增强（P＜0.05）。二氢杨梅素作用绒癌JEG-3及JAR细胞后,显著抑制细胞迁移能力,且具有浓度依赖性（P＜0.05）。不同浓度二氢杨梅素作用JEG-3和JAR细胞后,MMP-2 的mRNA和蛋白表达水平明显下降（P＜0.05）。 结论 二氢杨梅素能够抑制JEG-3及JAR细胞的增殖能力且具有浓度依赖性,同时二氢杨梅素可能通过下调绒癌JEG-3及JAR细胞中MMP-2 mRNA和蛋白的表达,抑制绒癌细胞的侵袭迁移。     

流体剪切应力对晚期内皮祖细胞生物学功能的影响

目的 研究流体剪切应力处理对晚期内皮祖细胞（endothelial progenitor cells,EPCs）体外及体内生物学功能的影响。 方法 密度梯度离心法分离大鼠骨髓单核细胞,应用EGM-2MV进行体外培养。以3~4代的EPCs,即晚期EPCs为靶细胞,对其施以1.2 Pa剪切应力处理。采用EdU标记技术、黏附能力测定实验、改良的Boyden小室、Annexin V/PI、β 半乳糖苷酶检测法、Matrigel法、荧光定量RT PCR等方法分别检测剪切应力对晚期EPCs增殖、黏附、迁移、凋亡、衰老、体外成血管及VEGF mRNA表达等生物学功能的影响。应用大鼠颈动脉损伤模型及细胞原位移植等实验手段检测剪切应力预处理对晚期EPCs修复受损内皮的影响。结果1.2 Pa剪切应力处理可不同程度提高晚期EPCs的增殖、黏附、迁移及成血管能力（P＜0.01）,上调VEGF的基因表达,抑制晚期EPCs的衰老及凋亡（P＜0.01）;移植经剪切应力预处理的晚期EPCs可加速损伤内皮的修复,减缓内膜的增生。结论 流体剪切应力可改善晚期EPCs的功能活性,提高晚期EPCs修复损伤血管内皮的能力, 这为EPCs的临床应用及剪切应力介导的细胞疗法提供了实验依据。     

趋化因子-8对高糖环境下脂肪间充质干细胞迁移能力的影响

目的 探讨高糖环境下,趋化因子-8（CXCL-8）对人脂肪间充质干细胞（hADMSCs）迁移能力的影响和机制。方法 建立高糖环境模型;在高糖条件下,建立CXCL-8实验组、Akt抑制剂组和高糖对照组,在正常条件培养的hADMSCs为正常对照组;分别用细胞划痕实验、Transwell细胞小室实验检测CXCL-8对hADMSCs的迁移能力影响,并用Western blotting、ELISA实验检测Akt、信号转导和转录激活因子（STAT3）和血管内皮生长因子（VEGF）等蛋白表达。 结果 相对高糖对照组,CXCL-8实验组hADMSCs细胞划痕面积闭合率和Transwell细胞小室迁移率均增高（P＜0.01）;而Akt抑制剂组hADMSCs细胞划痕面积闭合率或Transwell细胞小室迁移率比CXCL-8实验组皆降低（P＜0.01）;CXCL-8实验组磷酸化Akt、mTOP、STAT3蛋白表达增高,CXCL-8实验组上清液的VEGF、表皮细胞生长因子 (EGF)含量明显增高（P＜0.01）;而Akt抑制剂组hADMSCs分泌能力下降（P＜0.01）。 结论 高糖环境下,CXCL-8通过Akt-STAT3通路促进间充质干细胞（MSCs)旁分泌VEGF等因子,促进MSCs迁移,对招募宿主细胞归巢,促进组织损伤修复具有重要意义。     

姜黄素衍生物T83对同源不同辐射抗性鼻咽癌细胞凋亡的作用

 目的：比较4-芳甲叉类姜黄素衍生物T83对同源不同辐射抗性鼻咽癌细胞凋亡的作用差异。方法：采用MTT法、Hoechst 33342染色法、流式细胞术、Western blotting和qRT-PCR 检测和比较T83对CNE-2R和CNE-2细胞活力、细胞凋亡及线粒体膜电位、细胞procaspase-3/procaspase-9/Cyt-C蛋白表达和细胞PTEN/Akt/p27 mRNA水平的影响。结果：T83可抑制CNE-2R细胞的活性（24 h、48 h和72 h的IC50分别为0.9、0.4和0.2 μmol/L）,其抑制作用明显优于对CNE-2细胞的作用（24 h、48 h和72 h的IC50分别为1.8、0.5和0.4 μmol/L;P＜0.05）;T83作用48 h后,CNE-2R和CNE-2细胞中均观察到染色质浓缩、边集和分割成块状;T83作用48 h后,CNE-2R细胞晚期凋亡率升高（1.57%→27.26%）,明显高于CNE-2细胞（1.74%→8.15%;P＜0.05）;T83作用36 h后,CNE-2R细胞线粒体膜电位下降百分率（87.71%）明显高于CNE-2细胞（50.47%;P＜0.05）;T83作用48 h后,CNE-2R细胞中procaspase-3和procaspase-9蛋白表达明显低于CNE-2细胞,CNE-2R中Cyt-C蛋白表达明显高于CNE-2细胞。T83作用24 h后,PTEN和p27 mRNA水平明显上调,而Akt mRNA水平明显下调（P＜0.05）,且CNE-2R细胞的变化更加显著。结论：与CNE-2细胞相比,T83能更加明显地抑制PTEN/Akt/p27信号转导通路,启动线粒体凋亡途径,加速诱导CNE-2R细胞凋亡。     

CD133和CD34在肝细胞癌血管生成拟态形成中的表达及意义

背景：在恶性肿瘤中血管生成拟态的形成过程与肿瘤干细胞有密切联系。 目的：分析肝癌干细胞标志物CD133和CD34在肝细胞癌血管生成拟态形成中的表达及意义。 方法：建立肝癌细胞HCC97H、SMMC7721和正常肝细胞L02三维培养体系,结合激光捕获显微切割技术分离形成血管生成拟态的肝癌细胞,分别利用RT-PCR和Western blot技术检测CD133和CD34表达水平。 结果与结论：三维培养条件下,肝癌细胞HCC97H细胞形成血管生成拟态,肝癌细胞SMMC7721以及正常肝细胞L02未形成血管生成拟态。形成血管生成拟态的肝癌细胞HCC97H中CD133、CD34在mRNA及蛋白表达水平上均高于未形成血管生成拟态的肝癌细胞SMMC7721和正常肝细胞L02(P < 0.05)。表明高侵袭性肝癌细胞在三维培养下形成血管生成拟态,而低侵袭性肝癌细胞及正常肝细胞不能形成血管生成拟态;肝癌细胞形成血管生成拟态的过程中与表达肝癌干细胞有关。     

23-羟基桦木酸体外抑制血管生成的实验研究

目的：探讨23-羟基桦木酸（23-HBA）对体外血管生成的抑制作用。方法：采用磺酰罗丹明B（SRB）法测定23-HBA对人毛细血管内皮细胞（HMEC）增殖、迁移和小管形成的影响；用免疫组化染色法检测HMEC中CD31表达的变化。结果：23-HBA体外可明显抑制HMEC增值（IC50为40．44mg／L）、迁移和小管的形成，且呈明显的剂量依赖性。23-HBA的浓度为10mg／L时、HMEC中CD31的表达明显降低。结论：23-HBA体外具有明显抑制血管生成的作用．有可能成为有效的血管生成抑制剂。     

血管内皮生长因子、Eph受体酪氨酸激酶A2、基质金属蛋白酶-2和-9在卵巢癌血管生成拟态中的作用

目的 探讨血管内皮生长因子(VEGF)、Eph受体酪氨酸激酶A2 (EphA2)、基质金属蛋白酶(MMP)-2和MMP-9在卵巢癌血管生成拟态中的作用.方法 收集临床和预后资料完整的卵巢癌组织标本84例,切片经明确诊断后进行CD31和过碘酸-雪夫 (PAS) 双重染色,证实肿瘤组织中存在血管生成拟态,行VEGF、EphA2、MMP-2和MMP-9 免疫组织化学染色,根据染色指数统计免疫组织化学染色结果.结果 有血管生成拟态组(36/84)和无血管生成拟态组卵巢癌组织(48/84)的VEGF、EphA2、MMP-9表达差异有统计学意义,有血管生成拟态组的卵巢癌细胞VEGF、EphA2、MMP-9表达明显高于无血管生成拟态组.但有血管生成拟态组和无血管生成拟态组患者的MMP-2表达差异无统计学意义.结论 在卵巢癌中血管生成拟态和内皮依赖性血管并存,VEGF、EphA2和MMP-9等参与了卵巢癌血管生成拟态的形成.检测VEGF、EphA2和MMP-9可作为预测卵巢癌预后的间接指标.     

Dual VEGF/PDGF knockdown suppresses vasculogenic mimicry formation in choroidal melanoma cells via the Wnt5a/β-catenin/AKT signaling pathway

ObjectiveThis study aimed to explore the effects of knocking down both vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) on vasculogenic mimicry (VM) formation in choroidal melanoma (CM) cells.MethodsCell counting Kit (CCK)?8, monoclonal formation, wound healing, transwell and flow cytometry assays were used to observe the cell effects in CM cell line, ocular choroidal melanoma-1 cells (OCM-1) with respect to proliferation, migration, invasion and apoptosis. Three-dimensional (3D) cultures were also used to characterize VM tube structural effects in OCM-1 cells and western blotting was used to characterize protein expression changes in VM-related markers.ResultsDual VEGF/PDGF knockdown suppressed cell proliferation, migration and invasion, but promoted cell apoptosis. It also reduced VM tube structures in OCM-1 cells. VM associated markers including, VE-cadherin, EphA2 and MT1-MMP were also down-regulated in OCM-1 cells. Similarly, Wnt5a, β-catenin and phosphorylated-AKT levels were also down-regulated. Western blotting and 3D cultures further demonstrated that combined Wnt5a silencing with dual VEGF/PDGF knockdown significantly decreased VE-cadherin and EphA2 levels and reduced VM tube structures in OCM-1 cells.ConclusionsDual VEGF/PDGF knockdown suppressed cell growth and metastasis in OCM-1 cells, and blocked the Wnt5a/β-catenin/AKT signaling pathway thereby inhibiting VM formation.     

Overexpression of carcinoma and embryonic cytotrophoblast cell-specific Mig-7 induces invasion and vessel-like structure formation

Molecular requirements for carcinoma cell interactions with the microenvironment are critical for disease progression but are poorly understood. Integrin alpha v beta 5, which senses the extracellular matrix, is important for carcinoma cell dissemination in vivo. alpha v beta 5 signaling induces Mig-7, a novel human gene product that is apparently carcinoma-specific. We hypothesized that Mig-7 expression facilitates tumor cell dissemination by increasing invasion and vasculogenic mimicry. Results show that embryonic cytotrophoblasts up-regulated Mig-7 expression before they acquired an invasive phenotype capable of pseudovasculogenesis. Mig-7 protein primarily co-localized with vasculogenic mimicry markers factor VIII-associated antigen, vascular endothelial-cadherin, and laminin 5 gamma 2 chain domain III fragment in lymph node metastases. Overexpression of Mig-7 increased gamma 2 chain domain III fragments known to contain epidermal growth factor (EGF)-like repeats that can activate EGF receptor. Interestingly, EGF also induced Mig-7 expression. Carcinoma cell adhesion to laminins was significantly reduced by Mig-7 expression. Remarkably, in two-dimensional and three-dimensional Matrigel cultures, Mig-7 expression caused invasion and vessel-like structures. Melanoma cells, which were previously characterized to invade aggressively and to undergo vasculogenic mimicry, expressed Mig-7. Taken together, these data suggest that Mig-7 expression allows cells to sense their environment, to invade, and to form vessel-like structures through a novel relationship with laminin 5 gamma 2 chain domain III fragments.     

Molecular determinants of ovarian cancer plasticity

During development, the formation and remodeling of primary vascular networks occurs by vasculogenesis and angiogenesis. Recently, the term "vasculogenic mimicry" has been used by our laboratory and collaborators to reflect the embryonic-like ability of aggressive, but not nonaggressive, melanoma tumor cells to form a pattern of matrix-rich networks (containing channels) surrounding spheroids of tumor cells in three-dimensional culture, concomitant with their expression of vascular cell markers. Ovarian cancer is usually diagnosed as advanced stage disease in most patients when widespread metastases have already been established within the peritoneal cavity. In this study, we explored whether invasive ovarian carcinoma cells could engage in molecular vasculogenic mimicry reflected by their plasticity, compared with their normal cell counterparts. The data revealed that the invasive ovarian cancer cells, but not normal ovarian surface epithelial cells, formed patterned networks containing solid and hollow matrix channels when grown in three-dimensional cultures containing Matrigel or type I collagen, in the absence of endothelial cells or fibroblasts. Immunohistochemical analysis showed that matrix metalloproteinases (MMP)-1, -2, and -9, and MT1-MMP were discretely localized to these networks, and the formation of the networks was inhibited by treatment with MMP inhibitors. Furthermore, the RNase protection assay revealed the expression of multiple vascular cell-associated markers by the invasive ovarian cancer cells. In patient tumor sections from high-stage, high-grade ovarian cancers, 7 to 10% of channels containing red blood cells were lined by tumor cells. By comparison, all vascular areas in benign tumors and low-stage cancers were endothelial lined. These results may offer new insights and molecular markers for consideration in ovarian cancer diagnosis and treatment strategies based on molecular vascular mimicry by aggressive tumor cells.     

Tumor cell plasticity in uveal melanoma: microenvironment directed dampening of the invasive and metastatic genotype and phenotype accompanies the generation of vasculogenic mimicry patterns

The histological detection of laminin-rich vasculogenic mimicry patterns in human primary uveal melanomas is associated with death from metastases. We therefore hypothesized that highly invasive uveal melanoma cells forming vasculogenic mimicry patterns after exposure to a laminin-rich three-dimensional microenvironment would differentially express genes associated with invasive and metastatic behavior. However, we discovered that genes associated with differentiation (GDF15 and ATF3) and suppression of proliferation (CDKNa1/p21) were up-regulated in highly invasive uveal melanoma cells forming vasculogenic mimicry patterns, and genes associated with promotion of invasive and metastatic behavior such as CD44, CCNE2 (cyclin E2), THBS1 (thrombospondin 1), and CSPG2 (chondroitin sulfate proteoglycan; versican) were down-regulated. After forming vasculogenic mimicry patterns, uveal melanoma cells invaded only short distances, failed to replicate, and changed morphologically from the invasive epithelioid to the indolent spindle A phenotype. In human tissue samples, uveal melanoma cells within vasculogenic mimicry patterns assumed the spindle A morphology, and the expression of Ki67 was significantly reduced in adjacent melanoma cells. Thus, the generation of vasculogenic mimicry patterns is accompanied by dampening of the invasive and metastatic uveal melanoma genotype and phenotype and underscores the plasticity of these cells in response to cues from the microenvironment.     

GM-CSF对诱导人血管内皮细胞血管形成的影响及VEGF的作用

目的： 研究炎性因子粒细胞-巨噬细胞集落刺激因子（GM-CSF）对诱导人血管内皮细胞血管形成的影响及血管内皮生长因子（VEGF）的作用，为探讨GM-CSF和VEGF与动脉粥样硬化斑块内血管新生及斑块稳定性之间的关系提供实验基础。方法： 在 Matrigel上诱导人脐带静脉内皮细胞（HUVECs）形成管腔结构，从而建立稳定的血管形成的体外培养体系，然后施加实验因素。分别检测rhGM-CSF的浓度效应和时间效应，及加入VEGF165的作用。然后各实验组用CD34免疫细胞化学染色，光学显微镜下计数各组管腔数。最后用统计学方法进行分析。结果： 应用Matrigel可在体外诱导培养的HUVECs形成管腔结构。rhGM-CSF作用后，培养体系的管腔数逐渐增多，呈剂量及时间依赖效应；加入VEGF165后，管腔数显著增多。结论： 应用Matrigel可在体外诱导培养的HUVECs形成管腔结构。GM-CSF可以促进体外诱导人血管内皮细胞血管形成，并在一定范围内呈剂量及时间依赖效应。VEGF可以促进体外诱导人血管内皮细胞血管形成。