EGFL7与肿瘤研究新进展

EGFL7蛋白为一种内皮细胞特异性分泌因子,它是血管管腔形成所必需的因子,它的缺乏将导致管腔形成受阻,从而影响血管功能的完善.其在早期胚胎的血管中有较强的表达,而在成年人仅在少数器官(如:心脏、肺脏、肾脏)和肿瘤、炎症组织中有高水平表达.在肿瘤的生长转移过程中新生血管的作用十分重大,阻断肿瘤新生血管EGFL7的表达将有助于抑制肿瘤生长和转移,为肿瘤治疗提供一个新的途径.     

血管内皮细胞生长因子受体Flt-1结合小肽的融合表达及活性鉴定

血管内皮细胞生长因子 (VEGF)通过结合其酪氨酸激酶受体KDR、fms样酪氨酸激酶 1(Flt 1)调节新生血管形成 ;筛选能封闭VEGF结合Flt 1的小肽 ,可以通过阻断肿瘤血管形成 ,抑制实体瘤生长 .将从噬菌体 12肽库中筛选获得的 2个能与Flt 1结合的阳性噬菌体克隆 (F5 6和F90 )十二肽DNA(36bp)克隆到表达载体pQE4 2中 ,在大肠杆菌M15中稳定表达二氢叶酸还原酶融合蛋白(DHFR F5 6 F90 ) ,经变性、复性后得到纯度达 90 %的可溶性蛋白 .ELISA检测表明 ,DHFR F5 6 F90能结合可溶性受体sFlt 1和血管内皮细胞 ;12 5I VEGF竞争抑制实验显示 ,DHFR F5 6能竞争抑制VEGF同可溶性受体sFlt 1结合 .结果提示 ,F5 6可能是VEGF受体Flt 1的有效拮抗剂 ,具有抗肿瘤新生血管形成的潜在应用前景     

胃癌组织中PTEN、VEGF表达与肿瘤新生血管形成

目的探讨胃癌组织中PTEN、vascular endothelial growthfactor（VEGF）基因表达及其与肿瘤侵袭转移的关系。方法用RT-PCR和免疫组化方法检测胃癌、淋巴结转移组织中PTEN、VEGF mRNA和蛋白表达;用CD34检测肿瘤细胞微血管数。结果PTEN和VEGF mRNA表达阳性率在正常胃黏膜为76.5%与0.0%、胃癌组织为30.9%与69.1%、淋巴结转移组织23.6%与74.5%;PTEN和VEGF蛋白阳性率在正常胃黏膜为76.5%与0.0%、胃癌组织27.9%与82.4%、淋巴结转移组织16.3%与91.0%;胃癌组织中新生血管呈浸润生长,以淋巴结转移组织中明显。胃癌组织PTEN mRNA和蛋白低于正常胃黏膜（P〈0.01）,VEGF高于正常胃黏膜（P〈0.01）,PTEN与VEGF表达负相关（P〈0.05）,VEGF表达与新生血管形成正相关（P〈0.05）。结论PTEN基因失活和VEGF的过表达与新生血管形成相关,可能是通过调节包括VEGF在内的血管生成因子而在血管形成中起作用。     

小干扰RNA靶向VEGF基因体内外抑制乳腺癌细胞MCF-7的增殖

 血管生成与肿瘤生长、侵袭、转移密切相关.血管内皮生长因子能特异地促进内皮细胞分裂、增殖及迁移,在肿瘤新生血管生成过程中起着至关重要的作用.通过RNAi抑制VEGF表达的抗血管生成疗法可有效应用于肿瘤治疗.本研究采用化学修饰的siRNA在体内外抑制VEGF基因表达,探讨化学修饰的siRNA介导的RNA干扰技术在乳腺癌基因治疗的可行性和特异性.选用阳离子脂质体Lipofectamine^TM2000作为转染试剂,将针对人VEGF基因的小干扰RNA(small interfering RNA,siRNA)转染人类乳腺细胞株MCF-7和裸鼠移植瘤,在体内外诱导RNAi.采用四甲基偶氮唑蓝（MTT）法,逆转录聚合酶链反应(RT-PCR),蛋白印迹实验等检测siRNA治疗组和对照组VEGF基因表达及细胞增殖变化.体外实验结果显示：靶向VEGF基因siRNA转染乳腺癌MCF-7细胞后,细胞生长抑制率达52.5%;VEGF的mRNA和蛋白表达水平显著降低（P<0.01）;裸鼠体内实验结果显示：siRNA治疗组瘤组织的增长受到明显抑制;RT-PCR结果同时表明治疗组VEGF表达下调.体内外对照组各指标无显著变化.化学修饰的siRNA介导的RNAi在体内外均能成功下调靶基因VEGF的表达,抑制MCF-7细胞增殖,是潜在的肿瘤治疗新方法.     

可溶型三聚体血管生长抑制因子Kringle5的克隆,表达,纯化及活性研究

血管生长抑制因子Kringle 5 是目前发现的抑制血管内皮细胞增殖和肿瘤生长的活性最强的纤溶酶原片段,特异性高而毒副作用小,在肿瘤的治疗方面具有潜在的巨大价值和广阔的应用前景。根据K5基因的序列设计PCR引物,通过PCR从已有的克隆载体扩增出人纤维蛋白溶酶原的K5部分基因,将K5基因克隆入原核表达载体pET15b,经序列测定,成功构建了pET15b-K5非融合表达载体。将重组载体导入大肠杆菌中IPTG诱导表达,SDS-PAGE分析目的蛋白主要以可溶形式存在于菌体中,破碎后上清通过阳离子交换层析,纯化获得纯度大于95%的目标蛋白,脱盐后对分子量测定推测形成了三聚体。通过鸡胚绒毛尿囊膜法证明蛋白产物对鸡胚绒毛尿囊膜血管增生有一定的抑制作用。     

VEGF受体KDR胞外区基因的克隆及其在昆虫细胞中的表达

VEGF(血管内皮细胞生长因子,Vascular Endothelial Growth Factor)是刺激内皮细胞增殖和新生血管形成的最重要因子,与多种实体瘤的生长和转移密切相关。应用其可溶性受体阻断它的病理作用是一个非常有前景的课题。将VEGF受体KDR胞外区前三个Loop 969碱基对的cDNA片段克隆到杆状病毒表达载体pFastBacI,与杆状病毒表达载体Bacmid同源重组后,转染昆虫细胞SF9,获得重组杆状病毒并证明了目的基因的高效表达。经Western blot证实表达产物的特异性。经ELISA和体外生物学活性检测表明表达产物可阻断VEGF的生物学活性,抑制鸡胚CAM血管的生长。     

小鼠天然可溶性VEGF受体基因sflt-1的克隆及鉴定

 可溶性血管内皮细胞生长因子受体 (sFlt 1 )与膜表面受体Flt 1竞争结合血管内皮细胞生长因子 (VEGF) ,并且与膜表面受体Flt 1及KDR形成异源二聚体 .完全阻断VEGF的生物学活性 ,除与sFlt 1的结合部位结构域有关外 ,还与整个蛋白质分子的高效分泌表达有关 .而蛋白质分子的高效分泌表达与蛋白质在细胞内高尔基体及内质网的加工密切相关 ,基因工程重组可溶性受体由于一些尚未明了的原因 ,往往不能高效表达 ,从而大大影响了其应用价值 .利用RT PCR技术从胚胎小鼠中扩增出天然可溶性VEGF受体基因sflt 1 ,克隆于pcDNA3载体中 ,在COS 7细胞中短暂表达 ;并克隆至pET42b载体中 ,经IPTG诱导后 ,可大量稳定表达与His Tag形成的融合蛋白 ,经HisNi柱纯化 ,可特异性结合VEGF .可溶性受体sFlt 1在肿瘤组织的高效表达可有效阻断新生血管的形成 ,从而为肿瘤的治疗探索一种方法 .     

血管内皮生长因子基因对兔髂动脉内膜损伤的组织形态变化过程的影响

探讨血管内皮细胞的特异丝裂原－血管内皮生长因子（VEGF）基因阻止血管内膜损伤后形成再狭窄的组织变化过程。建立球囊拉伤血管内膜的兔髂动脉模型,将携带VEGF目的基因的真核表达载体pcDNA3/VEGF经多聚赖氨酸处理的PTCA球囊导管导入拉伤的血管内膜。VEGF基因组拉伤2周时血管内壁有VEGF mRNA和蛋白的高表达。血管内膜内皮化较快。2周时即有许多血管内皮细胞呈岛状分布。4周时内膜基本恢复光滑。内膜平滑肌细胞增生明显减少,而对照组2周时血管内膜粗糙,基底膜暴露,拉伤后4周仍无内皮细胞再生,最后形成虫蚀样改变。血管中膜平滑肌细胞穿过内弹性膜进入内膜并大量增生,内膜增厚。VEGF基因定位导入血管内壁后。VEGF mRNA和蛋白高表达且发挥其生物学效应,内皮细胞岛状增生,加快内膜内皮化,减轻内膜增厚。     

骨桥蛋白RNA干扰对人U251胶质瘤细胞在裸鼠体内生长的影响

研究下调骨桥蛋白(osteopontin,OPN)对人U251胶质瘤细胞在裸鼠体内生长的影响并探讨其对胶质瘤生长、侵袭的可能机制.应用RNA干扰技术,将OPN基因的慢病毒干扰载体LV-OPNshRNA感染U251细胞.将对照和试验组U251细胞分别接种裸鼠,建立裸鼠荷瘤模型.3周后测量肿瘤的体积、瘤重并做肿瘤组织病理切片分析;利用RT-PCR和免疫印迹法检测OPN、尿激酶型纤维蛋白酶原激活物(uPA)、基质金属蛋白酶(MMP-2、MMP-9)的mRNA和蛋白表达;免疫组化法检测肿瘤组织微血管密度和血管内皮生长因子（VEGF）表达情况. 经OPN的RNA干扰后,能显著降低肿瘤组织OPN mRNA水平及蛋白表达,有效抑制肿瘤细胞生长及侵袭能力, 肿瘤体积及重量的减小有统计学意义(P<0.05).感染组uPA、MMP-2和MMP-9的mRNA和蛋白表达明显减少, 肿瘤组织的MVD值和VEGF的表达均显著降低.上述结果表明,抑制OPN的表达能明显抑制人U251胶质瘤细胞在裸鼠体内的生长和侵袭,OPN可能通过激活uPA、MMP-2和MMP-9等蛋白酶降解细胞外基质和促进肿瘤血管生成,参与胶质瘤的生长.     

促吞噬肽衍生物 T 肽对裸鼠术后残瘤 VEGF 表达的影响

目的：探讨促吞噬肽衍生物T肽抑制裸鼠术后残瘤生长的作用机理。方法：建立MCF-7乳腺癌裸鼠皮下移植瘤术后残瘤模型,观察8mg／kg剂量T肽对残余肿瘤组织的生长情况,并采用免疫组化和Western印迹检测给药后残瘤组织血管内皮生长因子（VEGF）的表达,采用RT-PCR检测不同T肽浓度对血管内皮细胞VEGF基因转录的影响。结果：T肽对MCF-7乳腺癌裸鼠皮下移植瘤术后残瘤生长表现出良好的抑制作用,按照瘤重得出的抑制率为68．2％,按照肿瘤体积得出的抑制率为67．6％,T肽给药组裸鼠残瘤组织中VEGF的表达量较空白对照组明显减少。血管内皮细胞中T肽呈剂量相关性抑制VEGF基因的转录。结论：T肽的抗癌作用与其抑制肿瘤微环境肿瘤组织和血管内皮细胞中VEGF的表达有密切联系,预示着T肽有潜力成为预防术后残瘤生长的抗癌新药。     

Vascular Integration of Endothelial Progenitors During Multistep Tumor Progression

Bone marrow-derived endothelial precursor cells contribute to tumor neovascularization. However, it is unclear when during progressive tumor growth circulating precursors are recruited into the preexisting vascular network, and how they home specifically into the tumor microenvironment. Here, we summarize recent findings from mouse models of multistage carcinogenesis, which reveal distinct phases of angiogenic activity. Only advanced tumors with a highly heterogeneous, sprouting vasculature recruite endothelial progenitors into neovessels. Surprisingly, during progressive tumor growth endothelial cells acquire new characteristics and secrete CC chemokines, a group of chemoattractants with adjacent cysteins, which play a dual role by enhancing neovascularization in an autocrine and endocrine fashion. Locally, chemokines stimulate endothelial proliferation; systemically, they guide chemokine receptor-positive circulating progenitors into the tumor bed. Subsequently, endothelial progenitors are truly integrated into the network of pre-existing vessel. This mechanism represents a novel concept where not the tumor itself, but endothelial cells as components of the tumor-induced stroma foster neovascularization in a self-amplifying loop.     

人血管生成抑制素抑瘤研究进展

肿瘤的发展和转移需要新生血管的形成。人血管生成抑制素是近年发现的能够专一性抑制血管内皮细胞的内皮抑制因子。大量研究表明,在体外用血管生成抑制素处理血管内皮细胞可以抑制新生血管的形成,在体内单独使用血管生成抑制素,或者将血管生成抑制素与其他物质如基质金属蛋白酶、尿激酶联合处理荷瘤小鼠,可以降低小鼠体内肿瘤组织新生血管密度,抑制肿瘤的生长和肿瘤细胞的迁移。简要综述了血管生成抑制素抑制肿瘤生长和转移及其作用机理。     

GCP-2/CXCL6 synergizes with other endothelial cell-derived chemokines in neutrophil mobilization and is associated with angiogenesis in gastrointestinal tumors

The precise role of chemokines in neovascularization during inflammation or tumor growth is not yet fully understood. We show here that the chemokines granulocyte chemotactic protein-2 (GCP-2/CXCL6), interleukin-8 (IL-8/CXCL8), and monocyte chemotactic protein-1 (MCP-1/CCL2) are co-induced in microvascular endothelial cells after stimulation with pro-inflammatory stimuli. In contrast with its weak proliferative effect on endothelial cells, GCP-2 synergized with MCP-1 in neutrophil chemotaxis. This synergy may represent a mechanism for tumor development and metastasis by providing efficient leukocyte infiltration in the absence of exogenous immune modulators. To mimic endothelial cell-derived GCP-2 in vivo, GCP-2 was intravenously injected and shown to provoke a dose-dependent systemic response, composed of an immediate granulopenia, followed by a profound granulocytosis. By immunohistochemistry, GCP-2 was further shown to be expressed by endothelial cells from human patients with gastrointestinal (GI) malignancies. GCP-2 staining correlated with leukocyte infiltration into the tumor and with the expression of the matrix metalloproteinase-9 (MMP-9/gelatinase B). Together with previous findings, these data suggest that the production of GCP-2 by endothelial cells within the tumor can contribute to tumor development through neovascularization due to endothelial cell chemotaxis and to tumor cell invasion and metastasis by attracting and activating neutrophils loaded with proteases that promote matrix degradation.     

G-CSF stimulates angiogenesis and promotes tumor growth: potential contribution of bone marrow-derived endothelial progenitor cells

Solid tumors require neovascularization for their growth. Recent evidence indicates that bone marrow-derived endothelial progenitor cells (EPCs) contribute to tumor angiogenesis. We show here that granulocyte colony-stimulating factor (G-CSF) markedly promotes growth of the colon cancer inoculated into the subcutaneous space of mice, whereas G-CSF had no effect on cancer cell proliferation in vitro. The accelerated tumor growth was associated with enhancement of neovascularization in the tumor. We found that bone marrow-derived cells participated in new blood vessel formation in tumor. Our findings suggest that G-CSF may have potential to promote tumor growth, at least in part, by stimulating angiogenesis in which bone marrow-derived EPCs play a role.     

The involvement of endothelial progenitor cells in tumor angiogenesis

Endothelial progenitor cells (EPCs) have been isolated from peripheral blood CD34, VEGFR-2, or AC 133 (CD133) antigen-positive cells, which may home to site of neovascularization and differentiate into endothelial cells in situ. Endothelial cells contribute to tumor angiogenesis, and can originate from sprouting or co-option of neighbouring pre-existing vessels. Emerging evidence indicate that bone marrow-derived circulating EPCs can contribute to tumor angiogenesis and growth of certain tumors. This review article will summarize the literature data concerning this new role played by EPCs in tumor angiogenesis.     

Nicotine enhances neovascularization and promotes tumor growth

Solid tumors require vascularization for their growth. Bone marrow-derived endothelial progenitor cells participate in tumor angiogenesis. Here, we show that nicotine markedly accelerated growth of colon cancer cells inoculated subcutaneously in mice but had no effect on proliferation of carcinoma cells in vitro. We found that the tumor growth was associated with increased vascularization of the tumor and that bone marrow-derived cells contributed to the formation of the new blood vessels. Our findings show that nicotine promotes tumor growth, at least in part, by stimulating tumor-associated neovascularization.     

Inhibition of prostate tumor angiogenesis by the tumor suppressor CEACAM1

We have previously shown that CEACAM1, a cell-adhesion molecule, acts as a tumor suppressor in prostate carcinoma. Expression of CEACAM1 in prostate cancer cells suppresses their growth in vivo. However, CEACAM1 has no effect on the growth of prostate cancer cells in vitro. This difference suggests that the antitumor effect of CEACAM1 may be due to inhibition of tumor angiogenesis, perhaps by increased secretion of antiangiogenic molecules from the cells. In this study, we have demonstrated that expression of CEACAM1 in DU145 prostate cancer cells induced the production of a factor or factors that specifically blocked the growth of endothelial but not epithelial cells. Conditioned medium from the CEACAM1-expressing cells but not control luciferase-expressing cells inhibited endothelial cell migration up a gradient of stimulatory vascular endothelial growth factor in vitro and inhibited corneal neovascularization induced by basic fibroblast growth factor in vivo. Moreover, conditioned medium from CEACAM1-expressing cells induced endothelial cell apoptosis in vitro. Only medium conditioned by CEACAM1 mutants that were able to suppress tumor growth in vivo could cause endothelial cell apoptosis. These observations suggest that CEACAM1-mediated tumor suppression in vivo is, at least in part, due to the ability of CEACAM1 to inhibit tumor angiogenesis.     

Mesenchymal stromal cells promote tumor growth through the enhancement of neovascularization

Mesenchymal stromal cells (MSCs), also called mesenchymal stem cells, migrate and function as stromal cells in tumor tissues. The effects of MSCs on tumor growth are controversial. In this study, we showed that MSCs increase proliferation of tumor cells in vitro and promote tumor growth in vivo. We also further analyzed the mechanisms that underlie these effects. For use in in vitro and in vivo experiments, we established a bone marrow-derived mesenchymal stromal cell line from cells isolated in C57BL/6 mice. Effects of murine MSCs on tumor cell proliferation in vitro were analyzed in a coculture model with B16-LacZ cells. Both coculture with MSCs and treatment with MSC-conditioned media led to enhanced growth of B16-LacZ cells, although the magnitude of growth stimulation in cocultured cells was greater than that of cells treated with conditioned media. Co-injection of B16-LacZ cells and MSCs into syngeneic mice led to increased tumor size compared with injection of B16-LacZ cells alone. Identical experiments using Lewis lung carcinoma (LLC) cells instead of B16-LacZ cells yielded similar results. Consistent with a role for neovascularization in MSC-mediated tumor growth, tumor vessel area was greater in tumors resulting from co-injection of B16-LacZ cells or LLCs with MSCs than in tumors induced by injection of cancer cells alone. Co-injected MSCs directly supported the tumor vasculature by localizing close to vascular walls and by expressing an endothelial marker. Furthermore, secretion of leukemia inhibitory factor, macrophage colony-stimulating factor, macrophage inflammatory protein-2 and vascular endothelial growth factor was increased in cocultures of MSCs and B16-LacZ cells compared with B16-LacZ cells alone. Together, these results indicate that MSCs promote tumor growth both in vitro and in vivo and suggest that tumor promotion in vivo may be attributable in part to enhanced angiogenesis.     

Tumor cell-mediated neovascularization and lymphangiogenesis contrive tumor progression and cancer metastasis

Robust neovascularization and lymphangiogenesis have been found in a variety of aggressive and metastatic tumors. Endothelial sprouting angiogenesis is generally considered to be the major mechanism by which new vasculature forms in tumors. However, increasing evidence shows that tumor vasculature is not solely composed of endothelial cells (ECs). Some tumor cells acquire processes similar to embryonic vasculogenesis and produce new vasculature through vasculogenic mimicry, trans-differentiation of tumor cells into tumor ECs, and tumor cell–EC vascular co-option. In addition, tumor cells secrete various vasculogenic factors that induce sprouting angiogenesis and lymphangiogenesis. Vasculogenic tumor cells actively participate in the formation of vascular cancer stem cell niche and a premetastatic niche. Therefore, tumor cell-mediated neovascularization and lymphangiogenesis are closely associated with tumor progression, cancer metastasis, and poor prognosis. Vasculogenic tumor cells have emerged as key players in tumor neovascularization and lymphangiogenesis and play pivotal roles in tumor progression and cancer metastasis. However, the mechanisms underlying tumor cell-mediated vascularity as they relate to tumor progression and cancer metastasis remain unclear. Increasing data have shown that various intrinsic and extrinsic factors activate oncogenes and vasculogenic genes, enhance vasculogenic signaling pathways, and trigger tumor neovascularization and lymphangiogenesis. Collectively, tumor cells are the instigators of neovascularization. Therefore, targeting vasculogenic tumor cells, genes, and signaling pathways will open new avenues for anti-tumor vasculogenic and metastatic drug discovery. Dual targeting of endothelial sprouting angiogenesis and tumor cell-mediated neovascularization and lymphangiogenesis may overcome current clinical problems with anti-angiogenic therapy, resulting in significantly improved anti-angiogenesis and anti-cancer therapies.     

An angiogenic growth factor is expressed in human glioma cells.

Progression to increased malignancy frequently occurs in human brain tumors of glial origin and usually involves neovascularization--a massive proliferation of endothelial cells into the tumor tissue. We have shown previously that subversion of a normal growth factor-related pathway is frequently associated with human gliomas. Here we show that human glioma cell lines express the gene encoding the angiogenic peptide endothelial cell growth factor (ECGF) or acidic fibroblast growth factor (a-FGF) and that an ECGF-like polypeptide is produced by these cells. The glioma-derived growth factor was partially purified from cell extracts by heparin-Sepharose affinity chromatography where it eluted at 1.5 M sodium chloride. On reversed-phase h.p.l.c., growth factor activity for endothelial cells was eluted at the same concentration of acetonitrile as found for bovine brain-ECGF, also a potent mitogen for endothelial cells. Moreover, human glioma cells possess specific cell surface receptors for ECGF and are mitogenically stimulated by exogenous addition of this growth factor. Glioma derived-ECGF may therefore have a dual influence: first, by autocrine growth-stimulation of human gliomas and, second, by paracrine-stimulation of endothelial cell proliferation which results in neovascularization of the tumor tissue.