CXCR4单克隆抗体抑制乳腺癌MCF-7细胞裸鼠移植瘤的实验研究

目的：研究CXC趋化因子受体4（CXC chemokine receptor 4,CXCR4）单克隆抗体（CXCR4 mAb）对人乳腺癌MCF-7细胞裸鼠皮下移植瘤生长的影响,并初步探讨CXCR4 mAb抗肿瘤的作用机制。方法：采用8周龄Balb/c雌性裸鼠,建立乳腺癌MCF-7细胞裸鼠皮下移植瘤模型。运用CXCR4 mAb进行干预,从整体水平观察CXCR4 mAb对肿瘤生长的影响,采用免疫组织化学法检测肿瘤组织中增殖细胞核抗原（PCNA）、半胱氨酸天冬氨酸酶3（Caspase-3）和血管内皮细胞生长因子（VEGF）的表达情况。结果：CXCR4 mAb可明显抑制移植瘤的生长,瘤体抑制率达到71.4%;CXCR4 mAb治疗后的肿瘤组织中PCNA和VEGF表达明显下降,而Caspase-3表达上升。结论：CXCR4 mAb可能是通过抑制肿瘤细胞增殖、促进肿瘤细胞凋亡及抑制肿瘤血管形成而发挥抗肿瘤生长的作用。     

CXCR4单克隆抗体抑制乳腺癌MCF-7细胞裸鼠移植瘤的实验研究

目的 研究CXC趋化因子受体4(CXC chemokine receptor 4,CXCR4)单克隆抗体(CXCR4 mAb)对人乳腺癌MCF-7细胞裸鼠皮下移植瘤生长的影响,并初步探讨CXCR4 mAb抗肿瘤的作用机制.方法 采用8周龄Balb/c雌性裸鼠,建立乳腺癌MCF-7细胞裸鼠皮下移植瘤模型.运用CXCR4 mAb进行干预,从整体水平观察CXCR4 mAb对肿瘤生长的影响,采用免疫组织化学法检测肿瘤组织中增殖细胞核抗原(PCNA)、半胱氨酸天冬氨酸酶3(Caspase-3)和血管内皮细胞生长因子(VEGF)的表达情况.结果 CXCR4 mAb可明显抑制移植瘤的生长,瘤体抑制率达到71.4%;CXCR4 mAb治疗后的肿瘤组织中PCNA和VEGF表达明显下降,而Caspase-3表达上升.结论 CXCR4 mAb可能是通过抑制肿瘤细胞增殖、促进肿瘤细胞凋亡及抑制肿瘤血管形成而发挥抗肿瘤生长的作用.     

miR-206抑制 SDF-1/CXCR4信号活化诱导的乳腺癌细胞迁移和增殖

目的：研究 miR-206在乳腺癌细胞中的作用及其机制。方法 MDA-MB-231细胞分别转染miRNA-NC和 miR-206 mimic 后,荧光显微镜观察转染效率。同时,细胞添加不同剂量（20 ng·mL -1和40 ng·mL -1）基质细胞衍生因子1（SDF-1）处理,利用 Transwell 法检测细胞迁移,噻唑蓝（MTT）法检测细胞增殖,qRT-PCR 法检测细胞中 CXC 趋化因子受体4（ CXCR4）mRNA 表达水平,Western blot 检测细胞中CXCR4蛋白表达水平。结果 miRNA-NC 组和 miR-206 mimic 组细胞转染效率分别为（83.4±6.3）％和（87.6±8.3）％。与对照组比较,SDF-1显著促进细胞迁移和增殖（P ﹤0.05）。miR-206 mimic 转染明显抑制细胞迁移和增殖（ P ﹤0.05）。SDF-1处理促进细胞中 CXCR4 mRNA 和蛋白的表达水平（ P ﹤0.05）。miR-206 mimic 转染则抑制 CXCR4蛋白表达水平（P ﹤0.05）,但不影响 CXCR4 mRNA 表达（P ﹥0.05）。结论 miR-206通过抑制 CXCR4表达拮抗 SDF-1诱导乳腺癌细胞迁移和增殖作用。     

Stathmin蛋白的研究进展

目的: 探讨趋化因子受体CXCR4表达水平对人肺癌细胞转移潜能的影响.方法: 采用RT-PCR,FACS检测趋化因子受体CXCR4在肺癌细胞株95C,95D细胞的表达.构建CXCR4正义和反义真核表达质粒,用脂质体法转染至95C和95D细胞内,通过G418筛选出稳定转染株.通过趋化和趋化侵袭实验测定转染前后细胞对基质衍生因子(SDF-1α)的迁移能力,通过明胶酶谱法检测MMP-2活性,通过FACS检测细胞对血管内皮细胞的黏附能力.结果: CXCR4正义和反义真核表达质粒pcDNA3-X4和pcDNA3-ASX4能明显上调或下调肺癌细胞表面CXCR4的表达,上调95C细胞表面CXCR4的表达可以增强其对SDF-1α的趋化反应性、MMP-2活性及其对血管内皮细胞的黏附能力.相反,下调95D细胞表面CXCR4的表达抑制了其对SDF-1α的趋化反应性、MMP-2活性及其对血管内皮细胞的黏附能力.结论: 上调或下调肺癌细胞表面CXCR4表达可以显著增强或抑制其转移潜能,提示CXCR4表达水平与肺癌细胞转移潜能有关.     

SDF-1/CXCR7轴与肿瘤的研究进展

基质细胞衍生因子-1（SDF-1）及其受体CXCR4所构成的SDF-1/CXCR4生物轴在肿瘤发生、发展及肿瘤血管新生中发挥重要作用。最近研究发现CXCR7也是SDF-1的受体,且与肿瘤密切相关。全文就SDF-1新受体CXCR7的生物学特征,SDF-1/CXCR7轴在肿瘤细胞中的表达,SDF-1/CXCR7轴与肿瘤细胞增殖和存活、肿瘤侵袭和转移以及肿瘤血管新生的关系作一综述。     

SDF-1/CXCR7在肝细胞癌中的表达及临床意义

目的：研究趋化因子SDF-1及其受体CXCR7在肝细胞癌组织中的表达,探讨其表达水平与临床病理特征的关系。方法：采用SP免疫组织化学法检测55例肝细胞癌标本及28例正常肝组织标本中SDF-1、CXCR7的表达。结果：SDF-1、CXCR7在正常肝组织表达均阴性,癌组织中二者阳性表达率分别为63.6%和78.2%;SDF-1表达于癌细胞及间质上皮细胞,CXCR7主要表达于间质内皮细胞,SDF-1表达与门静脉癌栓有关,CXCR7表达与肝内病灶数目、门静脉癌栓有关。结论：SDF-1/CXCR7生物学轴通过促进新生血管生成,参与肝细胞癌的侵袭和转移。有望成为判断肝细胞癌侵袭性及预后的指标。     

CXCR4/SDF-1 轴调节人肺腺癌PC-9 细胞对Bends细胞血脑屏障模型功能的影响

目的：通过建立体外血脑屏障（blood brain barrier,BBB）模型,探讨人肺腺癌PC-9 细胞在CXCR4/SDF-1 轴作用下对BBB紧密连接蛋白的影响。方法：利用永生化的小鼠脑微血管内皮细胞Bends 进行单层培养,建立体外BBB模型;通过跨内皮细胞电阻（transendothelial electrical resistance,TEER）测定及荧光素钠通透性实验判定体外BBB模型的功能状态以及观察PC-9细胞对体外BBB 模型功能的影响。Western blotting 检测PC-9 细胞在CXCR4 抑制剂AMD3100、SDF-1 单独或联合（1 μg/ml AMD3100,100 ng/ml SDF-1,AMD3100+SDF-1）作用下对BBB模型功能和内皮细胞紧密连接蛋白表达的影响,Transwell 迁移实验检测CXCR4/SDF-1 轴对PC-9 细胞跨BBB模型细胞层迁移能力的影响。结果：Bends 细胞单层培养可形成紧密连接的“屏障”并产生较高的TEER,第96 h 达到（182.13±5.19）Ω·cm2;同时行荧光色钠通透性实验结果显示,BBB具有良好屏障性能,其通透率低于空白对照组（P<0.05）。PC-9 细胞作用后,BBB模型TEER逐渐降低,第24 h 降至（46.7±4.35）Ω·cm2;同时BBB 通透率较作用前显著提高（P<0.05）。PC-9 细胞在AMD3100 作用下能够上调内皮细胞紧密连接蛋白的表达（P<0.05）;AMD3100 处理组的PC-9 细胞穿过BBB的细胞数较空白组明显减少[ （43±2）vs（81±2）个,P<0.05]。结论：AMD3100 能够减弱PC-9 细胞对Bends 细胞建立的体外BBB模型紧密连接的破坏能力。     

SDF-1/CXCR4轴在白血病患者骨髓中的表达及与血管新生的关系

目的：探讨SDF-1/CXCR4轴在白血病患者骨髓中的表达及与血管新生的关系。方法:采用 CD45/SSC设门四色流式细胞仪检测55例白血病患者骨髓的CXCR4表达。采用ELISA法检测SDF-1/VEGF;采用Envinson二步法检测骨髓组织MVD;多重PCR法检测白血病患者IgH、TCRγ链V区和J区,采用G-显带技术进行细胞遗传学核型分析。结果:AML组SDF-1(2145.21±329.45)pg/ml、CXCR4阳性表达率 (60.01±18.5)％;ALL组SDF-1(2549.02±303.4）pg/ml、 CXCR4阳性表达率（70.22±12.73）％;CML组SDF-1(1929.72±253.81)pg/ml、CXCR4阳性表达率（40.05±16.69）％;CRAL组SDF-1(2070.98±159.98)pg/mL、CXCR4阳性表达率(58.4±11.8)％;与对照组相比均明显增高,P<0.05。急淋SDF-1/CXCR4表达率高于其他白血病组;白血病SDF-1/CXCR4表达与相关因素的分析中发现,ALL与外周血WBC数量有相关性(r=0.534、 0.567, P<0.01),与急性白血病伴髓外浸润者有意义(P<0.05）;SDF-1/CXCR4 表达与白血病患者血小板计数、年龄、性别差异、骨髓增生程度、染色体改变、急性白血病骨髓细胞CD34+表达、ALL的IgH和TCRγ链V区和J区基因重排等因素无明显关系。白血病患者骨髓SDF-1、CXCR4、VEGF的相关系数为0.552, 0.553, 0.531,P<0.05。三者具有显著相关性。结论:白血病骨髓液中SDF-1含量及各群细胞CXCR4高表达,其中ALL表达最高;急性白血病缓解后依然高表达;SDF-1/CXCR4表达与ALL的外周血白细胞数量、有髓外浸润者有关,白血病中SDF-1/CXCR4、VEGF高表达并且相互之间存在有相关性,与白血病血管新生有关联。     

CXCR4/SDF-1α对宫颈癌HeLa细胞定向迁移及增殖的影响

 目的了解CXCR4在HeLa细胞的表达情况,并借助细胞培养评价CXCR4/SDF-1α对HeLa细胞定向迁移及增殖的影响。方法CXCR4 mAb免疫染色HeLa细胞。用Transwell侵袭转移模型评价HeLa细胞的迁移情况,其中,上室中加入预先用(或不用)CXCR4单抗预孵育的HeLa细胞,下室中加入含0~100ng/mlSDF-1α的培养基。为评价CXCR4、SDF-1α对HeLa细胞增殖的影响,将HeLa细胞接种于有(无)SDF-1α和(或)CXCR4的低血清环境72h。结果CXCR4在所有HeLa细胞上均有表达。HeLa细胞能顺SDF-1α浓度差定向迁移,且这一作用可被CXCR4 mAb拮抗。SDF-1α能促进He-La细胞在低血清环境中增殖。结论CXCR4/SDF-1α参与了HeLa细胞定向迁移的过程并影响其增殖。     

SDF-1/CXCR4对结直肠癌肝转移瘤表达乙酰肝素酶的影响

目的研究基质细胞衍生因子1（stromal cell derived factor-1,SDF-1）对高表达趋化因子受体CXCR4的结直肠癌Lovo细胞表达乙酰肝素酶（heparanase, HPA）的影响,并探讨 SDF-1/CXCR4轴对裸鼠结直肠癌肝转移瘤模型中乙酰肝素酶表达的影响。方法分别用SDF-1和SDF-1/CXCR4的拮抗剂AMD3100处理直肠癌 Lovo细胞,运用RT-PCR和免疫细胞化学技术检测结直肠癌细胞HPA 的表达;建立裸鼠结直肠癌肝转移瘤模型,检测AMD3100干预和非干预组转移瘤细胞中HPA 蛋白的表达。结果（1）结直肠癌Lovo细胞经不同浓度（50 ng/ml、100 ng/ml、200 ng/ml ）SDF-1处理后,HPA mRNA的表达量逐渐增高。用相同浓度SDF-1分别处理直肠癌Lovo细胞12h和24h后,其HPA的表达量随作用时间的延长而增高。在相同作用时间内,其HPA的表达量随SDF-1作用浓度（50 ng/ml、100 ng/ml 、200 ng/ml）的升高而增高。（2）SDF-1处理组HPA 的mRNA和蛋白表达水平均高于SDF-1+AMD3100处理组和对照组。（3）肝脏转移瘤中HPA的表达量随AMD3100治疗剂量的增高而降低。结论（1）SDF-1可刺激结直肠癌Lovo细胞HPA表达增加,并且这一效应与SDF-1的浓度和作用时间有关。（2）AMD3100能有效抑制SDF-1刺激结直肠癌细胞表达HPA,使得肿瘤细胞侵袭能力下降,从而起到抑制肿瘤转移的作用。     

SDF-1 and CXCR4 are up-regulated by VEGF and contribute to glioma cell invasion

Glioma cells produce vascular endothelial growth factor (VEGF) to induce vascularization and thereby supply the malignant tissue with oxygen and nutrients. However, little is known about the direct effects of VEGF on tumor cells. In this study, we investigate the ability of VEGF to promote proliferation and invasion of human glioma cells (U251n). Since the chemokine and its receptor, SDF-1/CXCR4, promote glioma cell proliferation and are up-regulated in human glioblastomas, we also tested the effects of VEGF on SDF-1 and CXCR4 mRNA expression. Using cell culture, the effect of VEGF on proliferation of U251n cells was measured using ELISA to detect incorporated BrdU as a marker of DNA syntheses. The effects of VEGF and SDF-1 on U251n cell invasion and proliferation were measured using inhibitors to VEGF receptor1 and receptor2, DC101 and MF1, respectively, and a CXCR4 antagonist (AMD3100). SDF-1 and CXCR4 mRNA expression in U251n and U87MG cells were measured using quantitative PCR. VEGF antisense phosphorothioate oligodeoxynucleotide (AS-VEGF) was also used to down-regulate VEGF expression in U251n cells. VEGF significantly increased U251n cell proliferation and invasion in a dose-dependent manner. These effects were blocked by the VEGF receptor inhibitors, DC101/MF1. The CXCR4 antagonist AMD3100 blocked U251n increased invasion, but not proliferation. CXCR4 and SDF-1 mRNA were up-regulated when U251n and U87MG cells were treated with VEGF. Eight micrometer VEGF antisense phosphorothioate oligodeoxynucleotide (AS-VEGF) down-regulated CXCR4 and SDF-1 mRNA levels in U251n cells. VEGF has a direct effect on U251n glioma cell proliferation and invasion. VEGF up-regulates SDF-1 and CXCR4 mRNA expression, and contributes to U251n cell invasion.     

The anti-cancer compound Nordy inhibits CXCR4-mediated production of IL-8 and VEGF by malignant human glioma cells

The chemokine receptor CXCR4 plays an important role in tumor growth, angiogenesis and metastasis. Our previous studies showed that Nordy, a synthetic chiral compound of nordihydroguaiaretic acid, inhibited the growth and angiogenesis of various malignant tumors. In this study we examined the capacity of Nordy to regulate CXCR4–mediated production of angiogenic factors by human glioblastoma cells. We found that Nordy potently inhibited CXCR4 ligand SDF-1-induced production of IL-8 and vascular endothelial cell growth factor, two important angiogenic factors implicated in the progression of malignant tumors. Further study revealed that the effect of Nordy was attributable to its down-regulation of the expression of functional CXCR4 in glioblastoma cells. These results suggest that the anti-cancer activity of Nordy is due, at least in part, to its suppression of the chemokine receptor CXCR4 thus reducing the production of angiogenic factors by tumor cells. Y.-F. Ping, X.-H. Yao, and J.-H. Chen contributed equally to the study.     

Tumor stromal-derived factor-1 recruits vascular progenitors to mitotic neovasculature, where microenvironment influences their differentiated phenotypes

Mechanisms underlying tumor vasculogenesis, the homing and engraftment of bone marrow-derived vascular progenitors, remain undefined. We hypothesized that tumor cell-secreted factors regulate vasculogenesis. We studied vasculogenic and nonvasculogenic intracranial murine gliomas. A PCR screen identified stromal-derived factor-1 (SDF-1/CXCL12) and vascular endothelial growth factor (VEGF) expression by vasculogenic glioma cells and spontaneously arising vasculogenic tumors in NF1+/-:Trp53+/- mice, but not by nonvasculogenic glioma cells. Enforced SDF-1, not VEGF, expression in nonvasculogenic cells caused vasculogenesis. Combined SDF-1 and VEGF expression augmented vasculogenesis over SDF-1 expression alone. Blocking SDF-1 receptor CXCR4 reduced short-term homing and long-term engraftment of vascular progenitors. Implanting tumor cells secreting SDF-1 was therefore necessary and sufficient to incorporate marrow-derived precursors into tumor endothelium. SDF-1 seemed to exert these effects by acting locally intratumorally and did not cause an efflux of marrow-derived progenitors into circulation. Tumor microenvironment determined additional fates of marrow-derived cells. Hypoxia, observed with ectopic s.c. murine tumors at levels approximating that of intracranial human glioblastoma, interacted with tumor-secreted SDF-1 to expand engrafted vascular progenitor differentiated phenotypes to include pericytes as well as endothelium. In contrast, less hypoxic orthotopic intracranial murine gliomas contained only marrow-derived endothelium without marrow-derived pericytes. Furthermore, we found that vasculogenesis is significant for tumors because it generates endothelium with a higher mitotic index than endothelium derived from local sources. Although CXCR4 blockade selectively targeted endothelium generated by vasculogenesis, completely inhibiting vessel formation may require combination therapy targeting locally derived and marrow-derived endothelium.     

Immunohistochemical expression of stromal cell-derived factor-1 (SDF-1) and CXCR4 ligand receptor system in hepatocellular carcinoma

Angiogenesis is an essential process for progression of hepatocellular carcinoma (HCC). The alpha-chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 have been recognized for their roles in regulating neoangiogenesis. The role of SDF-1 and CXCR4 in HCC progression has been little analyzed. The study aims to evaluate immunohistochemical expression of the SDF-land CXCR4 in HCC tissue and non-HCC tissue. Formalin-fixed paraffin-embedded tissue sections of 28 HCC, 7 hepatocellular adenoma (HA), 26 cirrotic nodules (CN) and 16 normal liver tissues (NLT) were immunostained for SDF-1 and CXCR4. SDF-1 and CXCR4 are detected in sinusoidal endothelial cells in HCC tissue, and their expressions are significantly higher than in non-HCC tissues. There was no significant correlation between SDF-1 expression and CXCR4 protein and the grade and stage of HCC. Overexpressions of SDF-1 and CXCR4 in sinusoidal endothelial cells in HCC suggest that the SDF-1/CXCR4 pathway plays a possible role in HCC progression through neoangiogenesis. Our data suggest that molecular strategies aimed at inhibiting the CXCR4/SDF-1 pathway might be of therapeutic use for the treatment of HCC.     

Blockade of the stromal cell-derived factor-1/CXCR4 axis attenuates in vivo tumor growth by inhibiting angiogenesis in a vascular endothelial growth factor-independent manner

The interaction between the chemokine receptor CXCR4 and its specific ligand, stromal cell-derived factor-1 (SDF-1/CXCL12), mediates several cellular functions. In cancer, SDF-1-positive or CXCR4-positive cells of various lineages are detected within tumor tissues. Recent intensive research has indicated the possibility that blocking CXCR4 could reduce the metastatic potential of cancer cells. Here, we show that the inhibition of the SDF-1/CXCR4 axis decreases the growth of s.c. gastrointestinal tumors through the suppression of tumor neoangiogenesis. The neutralization of CXCR4 suppressed the growth in vivo of tumors derived from mouse Colon38 and PancO2 cells, whereas it did not affect the growth of Colon38 and PancO2 cells in vitro. This attenuation of tumor growth was found to be independent of the expression of CXCR4 by the cancer cells themselves, because CXCR4 knocked-down Colon38 cells grew similarly to control cells. Furthermore, CD31-positive tumor capillaries were reduced to 45% (P < 0.001) and intratumor blood flows were decreased to 65% (P < 0.01) by blockade of CXCR4. The vascular endothelial growth factor (VEGF) concentration in the tumors was not affected by the neutralization of CXCR4. Taken together with the detection of CXCR4-positive endothelial cells in the tumor tissues, the findings suggest that the antiangiogenic effects of the blockade of CXCR4 are related to a reduction of the establishment of tumor endothelium independently of VEGF inhibition. Our data indicate that the SDF-1/CXCR4 pathway might be a general target for anticancer strategies and that blocking this system could be cooperatively effective in combination with other antiangiogenic therapies, such as blockade of VEGF.     

基质细胞衍生因子-1及其受体CXCR4在白血病中表达的临床意义

 基质细胞衍生因子-1（SDF-1）是早期细胞生长因子,属趋化因子亚家族成员之一。其受体是CXCR4、SDF-1、CXCR4配体受体系统在血细胞的生成、干细胞归巢、血管新生以及白血病细胞的浸润等生理和病理过程中发挥重要作用。研究CXCR4在白血病中的表达及其与白血病浸润的关系,对丰富白血病形态学、免疫学、细胞遗传学（MIC）诊断分型的免疫学指标,为防治白血病浸润、复发而采取相关分子靶向治疗都具有重要意义。