真核表达载体p4CCL20-ZsGreen1-DR 的构建与鉴定

背景:抗炎药物高通量筛选体系的建立,可为相关药物的研究提供一个理想的技术平台.目的:构建以核因子B 顺式作用元件4×CCL20 基序为增强子,以SV40 为启动子,以ZsGreen1-DR 为报告基因的真核表达载体p4CCL20-ZsGreen1-DR.方法:以PGL2-control 质粒为模板,PCR 扩增目的片段SV40,两侧引入KpnⅠ/BamH Ⅰ酶切位点,克隆至pZsGreen1-DR 质粒的Kpn Ⅰ /BamH Ⅰ酶切位点中,构建成pSV40-ZsGreen1-DR 载体.将4×CCL20 基序双链DNA 克隆到pSV40-ZsGreen1-DR 载体的BglⅡ和EcoRⅠ酶切位点之间,构建p4CCL20-ZsGreen1-DR 重组质粒.结果与结论:经过DNA 测序分析证实p4CCL20-ZsGreen1-DR 重组质粒构建成功.该重组质粒可作为抗炎药物高通量筛选体系的基础.     

真核表达载体p4CCL20-ZsGreen1-DR的构建与鉴定（英文）

背景：抗炎药物高通量筛选体系的建立,可为相关药物的研究提供一个理想的技术平台。目的：构建以核因子κB顺式作用元件4×CCL20基序为增强子,以SV40为启动子,以ZsGreen1-DR为报告基因的真核表达载体p4CCL20-ZsGreen1-DR。方法：以PGL2-control质粒为模板,PCR扩增目的片段SV40,两侧引入KpnⅠ/BamHⅠ酶切位点,克隆至pZsGreen1-DR质粒的Kpn Ⅰ/BamH Ⅰ酶切位点中,构建成pSV40-ZsGreen1-DR载体。将4×CCL20基序双链DNA克隆到pSV40-ZsGreen1-DR载体的BglⅡ和EcoRⅠ酶切位点之间,构建p4CCL20-ZsGreen1-DR重组质粒。结果与结论：经过DNA测序分析证实p4CCL20-ZsGreen1-DR重组质粒构建成功。该重组质粒可作为抗炎药物高通量筛选体系的基础。     

CCL5-CCR5生物轴与EMT在乳腺癌转移中的相互作用

趋化因子配体5(CCL5)是趋化性细胞因子CC亚族成员之一,参与细胞增殖、炎性反应、变态反应及肿瘤的生长和转移等过程。CCL5与其受体(CCR5)组成的生物轴是乳腺癌侵袭及转移的重要调节机制。上皮-间质转化(EMT)是近来研究癌细胞浸润和转移的重要机制。本文对CCL5-CCR5生物轴与EMT在乳腺癌转移中的最新进展进行综述。     

Chemoattractant Signaling between Tumor Cells and Macrophages Regulates Cancer Cell Migration, Metastasis and Neovascularization

Tumor-associated macrophages are known to influence cancer progression by modulation of immune function, angiogenesis, and cell metastasis, however, little is known about the chemokine signaling networks that regulate this process. Utilizing CT26 colon cancer cells and RAW 264.7 macrophages as a model cellular system, we demonstrate that treatment of CT26 cells with RAW 264.7 conditioned medium induces cell migration, invasion and metastasis. Inflammatory gene microarray analysis indicated CT26-stimulated RAW 264.7 macrophages upregulate SDF-1α and VEGF, and that these cytokines contribute to CT26 migration in vitro. RAW 264.7 macrophages also showed a robust chemotactic response towards CT26-derived chemokines. In particular, microarray analysis and functional testing revealed CSF-1 as the major chemoattractant for RAW 264.7 macrophages. Interestingly, in the chick CAM model of cancer progression, RAW 264.7 macrophages localized specifically to the tumor periphery where they were found to increase CT26 tumor growth, microvascular density, vascular disruption, and lung metastasis, suggesting these cells home to actively invading areas of the tumor, but not the hypoxic core of the tumor mass. In support of these findings, hypoxic conditions down regulated CSF-1 production in several tumor cell lines and decreased RAW 264.7 macrophage migration in vitro. Together our findings suggest a model where normoxic tumor cells release CSF-1 to recruit macrophages to the tumor periphery where they secrete motility and angiogenic factors that facilitate tumor cell invasion and metastasis.     

CD4 Regulates Susceptibility to Fas ligand– and Tumor Necrosis Factor–mediated Apoptosis

The current knowledge of CD4 function is limited to its role as a necessary coreceptor in TCR-initiated signaling. We have investigated whether CD4 regulates additional T cell functions. Using human primary resting CD4⁺ T cells, we demonstrate that CD4 activation is sufficient to induce lymphocyte death. Immediately after CD4 cross-linking, CD4⁺ T cells are rendered susceptible to apoptosis mediated by TNF or FasL. This, together with the concomitant induction of FasL within the same population, results in significant CD4⁺ T cell death in vitro. The CD4-dependent induction of susceptibility to apoptosis that is mediated by TNF or FasL is protein synthesis independent but phosphorylation dependent. After CD4 activation, PKC regulates susceptibility to apoptosis mediated by FasL but not the induction of susceptibility to TNF-dependent apoptosis. Moreover, significant differences between CD3 and CD4 activation were observed with regards to the kinetics of induction of CD4⁺ T cell susceptibility to FasL- and TNF-mediated apoptosis. Altogether, these results provide a model with which to study the molecular mechanisms regulating lymphocyte survival after CD4 activation, and highlight the potential role of CD4 in controlling lymphocyte apoptosis under physiological conditions or in disease states such as HIV infection.     

CXCR2 and CXCR4 antagonistically regulate neutrophil trafficking from murine bone marrow

Neutrophils are a major component of the innate immune response. Their homeostasis is maintained, in part, by the regulated release of neutrophils from the bone marrow. Constitutive expression of the chemokine CXCL12 by bone marrow stromal cells provides a key retention signal for neutrophils in the bone marrow through activation of its receptor, CXCR4. Attenuation of CXCR4 signaling leads to entry of neutrophils into the circulation through unknown mechanisms. We investigated the role of CXCR2-binding ELR⁺ chemokines in neutrophil trafficking using mouse mixed bone marrow chimeras reconstituted with Cxcr2^–/– and WT cells. In this context, neutrophils lacking CXCR2 were preferentially retained in the bone marrow, a phenotype resembling the congenital disorder myelokathexis, which is characterized by chronic neutropenia. Additionally, transient disruption of CXCR4 failed to mobilize Cxcr2^–/– neutrophils. However, neutrophils lacking both CXCR2 and CXCR4 displayed constitutive mobilization, showing that CXCR4 plays a dominant role in neutrophil trafficking. With regard to CXCR2 ligands, bone marrow endothelial cells and osteoblasts constitutively expressed the ELR⁺ chemokines CXCL1 and CXCL2, and CXCL2 expression was induced in endothelial cells during G-CSF–induced neutrophil mobilization. Collectively, these data suggest that CXCR2 signaling is a second chemokine axis that interacts antagonistically with CXCR4 to regulate neutrophil release from the bone marrow.     

骨髓增生异常综合征中SDF-1/CXCR4与细胞凋亡关系的研究

目的:探讨骨髓增生异常综合征(myelodysplastic syndromes,MDS)无效造血和高风险MDS向白血病转化的可能影响因素。方法:选取22例初发MDS患者(15例低危,7例高危)以及7例初发急性白血病患者,通过流式细胞仪检测其骨髓CD34+细胞的凋亡情况,同时以8例增生性贫血患者作为对照组。结果:发现在低危MDS组[国际预后评分系统(IPSS)评分≤1.0]骨髓内CD34+细胞的凋亡率明显高于高危MDS组(IPSS评分≥1.5)(21.33%比7.27%,P<0.01),同样低危MDS组骨髓内CD34+细胞的凋亡率明显高于白血病组(21.33%比7.53%,P     

High expression of CXCR4 and CXCR7 predicts poor survival in gallbladder cancer

Chemokine receptors play a prominent role in cancer progression and metastasis. This study investigated whether the expression of CXC chemokine receptor types 4 and 7 (CXCR4 and CXCR7, respectively), determined immunohistochemically, was associated with clinicopathological characteristics and postoperative survival in gallbladder cancer specimens from 72 patients. CXCR4 was detected in the cytoplasm and/or nucleus of gallbladder cancer cells, but CXCR7 was detected only in the cytoplasm. Expression of either CXCR7 or CXCR4 in the cytoplasm was associated with tumour stage. Expression of nuclear CXCR4 was associated with lymph node metastases and lymphatic invasion. Cytoplasmic expression of CXCR4 and CXCR7 were each independent risk factors for worse postoperative survival. Further research is required to clarify the mechanisms involved in these associations and to determine their potential prognostic and therapeutic implications.     

Delivery of siRNA Using CXCR4-targeted Nanoparticles Modulates Tumor Microenvironment and Achieves a Potent Antitumor Response in Liver Cancer

Antiangiogenic therapy has recently emerged as a highly promising therapeutic strategy for treating hepatocellular carcinoma (HCC). However, the only clinically approved systemic antiangiogenic agent for advanced HCC is sorafenib, which exerts considerable toxicity. Moreover, acquired resistance to antiangiogenic therapy often develops and restricts the therapeutic efficacy of this treatment. Hence, in this study, we develop a CXCR4-targeted lipid-based nanoparticle (NP) formulation to specifically deliver vascular endothelial growth factor (VEGF) siRNA as an antiangiogenic substance into HCC. AMD3100, a CXCR4 antagonist, is added into NPs to serve as both a targeting moiety and a sensitizer to antiangiogenic therapy. We demonstrate that AMD-modified NPs (AMD-NPs) can efficiently deliver VEGF siRNAs into HCC and downregulate VEGF expression in vitro and in vivo. Despite the upregulation of the SDF1α/CXCR4 axis upon the induction of hypoxia after antiangiogenic therapy, CXCR4 inhibition by AMD-NPs in combination with either conventional sorafenib treatment or VEGF siRNA prevents the infiltration of tumor-associated macrophages. These dual treatments also induce synergistic antiangiogenic effects and suppress local and distant tumor growth in HCC. In conclusion, the tumor-targeted multifunctional AMD-NPs that co-deliver VEGF siRNA and AMD3100 provide an effective approach for overcoming tumor evasion of antiangiogenic therapy, leading to delayed tumor progression in HCC.     

CCL20抑制小鼠胸腺CD4^＋CD25^＋天然调节性T细胞的发育

本研究探讨趋化因子CCL20对小鼠胸腺CD4^＋CD25^＋双阳性调节性T细胞发育的影响,为天然调节性T细胞调控的研究提供基础数据。采用14.5天龄胚鼠胸腺进行体外培养,以FACS检测不同时间点胸腺CD4^＋CD25^＋细胞的改变,同时计数每个胸腺小叶的细胞数变化。结果表明：体外胸腺培养的第1-6天胸腺细胞比例、细胞数量的变化趋势与胸腺体内发育的第14.5、15、16、17、18、19天CD4^＋CD25^＋双阳性T细胞的比例、细胞数量的变化趋势相似;在胸腺体外培养的第1-6天,CD4＋CD25^＋T细胞占CD4^＋T细胞的比例分别为58.29%、12.14%、6.08%、17.78%、9.06%、4.04%,占CD25^＋T细胞的比例分别为3.75%、10.81%、17.20%、51.93%、61.64%、80.06%,这一发育趋势与体内结果具有一致性。在4μg/ml的CCL20干预下,胸腺体外培养的第3、6天CD4^＋CD25^＋胸腺细胞分别从3.24±0.18、3.96±0.24下降至1.27±0.11、1.76±0.22（p值均〈0.001）。结论：体外培养的CD4^＋CD25^＋双阳性胸腺细胞数量和比例变化与体内发育变化趋势一致,CCL20明显下调胸腺CD4^＋CD25^＋的表达,这将为天然调节性T细胞的调控研究提供有效的参考依据。     

趋化因子受体-4在再生障碍性贫血中的表达及意义

目的检测趋化因子受体-4(CXCR4)在再生障碍性贫血患者骨髓细胞的表达,探讨再生障碍性贫血的发病机制。方法运用免疫组化方法检测20例确诊的再生障碍性贫血患者骨髓细胞中CXCR4的表达部位,运用光密度值分析CXCR4的蛋白含量。结果 (1)CXCR4阳性染色主要在细胞质,呈棕黄色表达;(2)CXCR4在再生障碍性贫血组的光密度值(0.259 8±0.050 7)明显高于对照组(0.074 0±0.009 7),差异具有统计学意义(t=15.69,P<0.001);(3)相关性分析提示再生障碍性贫血组CXCR4蛋白含量与外周血白细胞、血红蛋白、血小板呈负相关(相关系数r分别为-0.949、-0.865、-0.839,均P<0.001)。结论 CXCR4在再生障碍性贫血患者骨髓细胞的表达明显增加,且与外周血细胞呈负相关,CXCR4可能参与再生障碍性贫血的发病机制。     

趋化因子CCL21与冠心病相关性研究

目的探讨趋化因子CCL21与冠心病的关系。方法根据临床诊断标准及冠状动脉造影结果,将68例冠心病患者分为稳定型心绞痛组（22例）和急性冠状动脉综合征组（46例）,另取20例冠状动脉造影正常者为对照组;采用酶联免疫吸附测定法检测血清CCL21的水平,应用Gensini积分系统衡量冠状动脉病变程度。结果急性冠状动脉综合征组血清CCL2水平明显高于稳定型心绞痛组,而稳定型心绞痛组血清CCL21水平又明显高于对照组,（180.1±42.6）ng／L vs（145.3±33.7）ng／L vs（105.8±33.9）ng／L（均P〈0.01）;血清CCL21水平和冠状动脉Gensini积分呈显著正相关（r=0.466,P〈0.01）。结论血清CCL21水平可能反映了冠状动脉粥样硬化病变程度,并在斑块不稳定过程中起了重要作用。     

CCL20单克隆抗体与雷公藤治疗胶原诱导性关节炎模型小鼠的对照研究

目的 研究趋化因子CCL20在胶原诱导性关节炎(collagen-induced arthritis,CIA)小鼠中的表达,探讨CCL20单克隆抗体对CIA小鼠的治疗作用及临床治疗类风湿关节的潜在价值.方法 ①建立CIA小鼠模型:40只DBA/1小鼠随机分为健康对照组、模型组、CCL20单克隆抗体治疗组、雷公藤治疗组;②观察不同时间点小鼠一般情况、关节肿胀度;③测定造模42天后小鼠血清CCL20、肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)的表达水平;④观察各组小鼠关节病理变化.结果 ①模型组、雷公藤组和单抗组CCL20,TNF-α和IL-1β均高于对照组,CCL20(29.45±4.92) ng/L、(18.32±4.80) ng/L、(14.13±3.82) ng/L vs (2.83±1.37) ng/L;TNF-α(63.1±2.82) ng/L、(43.15±2.31) ng/L、(42.91±2.02) ng/L vs (26.78±3.50) ng/L;IL-1β(83.72±4.04) ng/L、(53.78±5.89) ng/L、(56.77±4.98)ng/L vs(16.54±2.45)ng/L,雷公藤组和单抗组的CCL20、TNF-α和IL-1β均低于模型组.②HE染色观察,模型组较正常组滑膜肥厚,排列紊乱、滑膜内有大量炎症细胞浸润,有血管翳形成.雷公藤组和单抗组较模型组明显减轻.结论 CCL20在CIA小鼠中异常高表达,以CCL20单克隆抗体阻断CCL20作用可有效缓解关节炎症,且与雷公藤效果相当,CCL20单克隆抗体在临床治疗类风湿关节炎方面具有潜在价值.     

趋化因子受体在嗜铬细胞瘤中的表达及意义

目的:通过检测趋化因子受体(CXCR4)在肾上腺良性嗜铬细胞瘤(BPCC)和恶性嗜铬细胞瘤(MPCC)中的表达,探讨诊断MPCC的辅助指标,为鉴别嗜铬细胞瘤(PCC)的生物学行为提供依据。方法:选取1996至2009年期间我院临床资料和病理资料完整的PCC手术标本141例(其中BPCC113例为良性组,MPCC28例为恶性组),另取10例因良性肾疾患行肾切除时获取的同侧正常肾上腺髓质作为对照组。采用免疫组化技术检测BPCC、MPCC及对照组中CXCR4的表达;同时随机选取60例标本,其中BPCC40例,MPCC20例,对患者的临床资料进行统计分析。结果:CXCR4在MPCC组中的表达率为85.71%,明显高于其在BPCC组中的表达(38.05%),两组间差异有统计学意义(P<0.05)。临床资料示MPCC组发病年龄明显低于BPCC组,MPCC平均发病年龄为38岁,BPCC平均发病年龄为45岁(P<0.05)。结论:CXCR4可作为MPCC的预判指标。对年龄较轻的PCC患者,应警惕其为恶性的可能。     

Antagonism between Epidermal Growth Factor and Phorbol Ester Tumor Promoters in Human Breast Cancer Cells

It has been suggested that the phorbol ester tumor promoters act via the receptor-effector system for epidermal growth factor (EGF), since they interact with the EGF receptor system and mimic many of the effects of EGF in cultured cells. We have studied the interaction of phorbol esters with the EGF-responsive MCF-7 human breast cancer cell line. Similar to other systems, phorbol esters inhibit EGF binding in MCF-7 cells in a manner paralleling their potency as tumor promoters in mice. The effect is specific for EGF since the membrane binding of insulin is unaffected. Like EGF, the potent phorbol ester 12-O-tetradecanoyl-13-phorbol acetate (TPA) stimulates protein synthesis as indicated by a twofold increase in [(3)H]leucine incorporation into protein after 24 h in TPA. Cell morphology, however, is significantly different with TPA treatment. After 24-48 h in TPA, cells become markedly enlarged with increased cytoplasmic vacuolization and increased membrane microvilli. This is reflected in a fourfold increase in the protein/DNA ratio (control 13.1; TPA 55.9). Furthermore, TPA inhibits cell division in media with or without serum, and prevents growth stimulation by EGF. Low TPA concentrations (1.0 ng/ml) are active, and 10 ng/ml results in maximal inhibition of cell replication. Other phorbol esters inhibit MCF-7 cells relative to their tumor promoting activity in vivo and their ability to inhibit EGF binding in these cells. After 24 h in TPA, incorporation of [(3)H]thymidine into DNA is markedly reduced and the thymidine labeling index falls (33% to 2%) indicating very few S-phase cells. Growth inhibition is reversible by removing TPA from the medium. Similar inhibitory effects are seen with the two other human breast cancer cell lines studied, ZR75-1 and MDA-MB-231. In conclusion, phorbol esters may interact with the EGF receptor domain in MCF-7 human breast cancer cells, but they have distinct effects on cell morphology and growth suggesting alternative pathways of action. The antineoplastic activity of these compounds needs further investigation.     

真核细胞翻译起始因子4E在NHL血管生成中的作用研究

目的探讨真核细胞翻译起始因子4E(EIF4E)和非霍奇金淋巴瘤(NHL)血管生成的关系。方法检测52例初诊NHL患者淋巴瘤石蜡切片EIF4E和平均微血管密度(MVD)的表达。构建反义EIF4E表达载体,电转染至Burkirt淋巴瘤细胞系Raji细胞,应用western-blot方法检测Raji细胞转染前后和正常人外周血淋巴细胞(NPBMC)中血管内皮生长因子(VEGF)的表达。结果在NHL患者中,侵袭性淋巴瘤EIF4E和MVD的表达明显高于惰性淋巴瘤的表达(P<0.05),且EIF4E与MVD的表达呈正相关(r=0.695,P<0.01)。成功构建EIF4E反义表达载体,将其转染入Raji细胞后,VEGF表达抑制。结论EIF4E在NHL的肿瘤血管生成中发挥作用,可能通过上调VEGF的表达而促进肿瘤血管生成。     

CXCR4在复发性胶质瘤中的表达及意义

[目的]探讨CXCR4作为脑胶质瘤恶性程度分级指标及治疗靶标的意义.[方法]采用免疫组化S-P法,检测复发前后人脑胶质瘤组织中CXCR4的表达.[结果]CXCR4在10例正常脑组织中未见表达.在31例原发性和复发性胶质瘤组织中,CXCR4阳性率分别为61.2%（19/31）和87.1%（27/31）,两者比较其差异有统计学意义（P〈0.05）.CXCR4阳性表达与人脑胶质瘤的肿块大小、vimentin表达不相关,而与肿瘤的病理分级、Ki-67表达正相关（P〈0.01,P〈0.05）.[结论]CXCR4与胶质瘤的恶性程度相关,可能作为胶质瘤恶性程度的分级指标;CXCR4在人脑胶质瘤组织中高表达,尤其在复发后阳性表达率显著增高,针对CXCR4为治疗靶点的脑胶质瘤分子靶向治疗可能是潜在的有力的治疗手段.     

Imaging CXCR4 Expression with Iodinated and Brominated Cyclam Derivatives

Molecular Imaging and Biology - CXCR4 is one of several “chemokine” receptors expressed on malignant tumors (including GBM and PCNSL) and hematopoietic stem cells. Although...