CXCR4对小鼠腹水型肝癌细胞淋巴转移潜能的影响

目的探讨趋化因子受体CXCR4表达水平对小鼠腹水型肝癌细胞淋巴转移潜能的影响。方法采用RT-PCR,流式细胞仪检测趋化因子受体CXCR4在小鼠腹水性肝癌细胞株Hca-F和Hca-P细胞的表达。通过归巢实验测定用抗体封闭CXCR4表达前后细胞特异性的向淋巴管迁移能力。结果CXCR4及其mRNA在高转移潜能小鼠肝癌细胞系Hca-F的表达高于低转移潜能小鼠肝癌细胞系Hca-P的表达。CX-CR4中和抗体能够抑制Hca-F细胞在体内向淋巴结转移。结论CXCR4在Hca-F、Hca-P细胞表面表达水平不同,可能是导致它们向淋巴管转移的潜能不同的影响因子之一,CXCR4表达水平与Hca-F、Hca-P细胞的特异性淋巴管转移潜能有关。     

重组人肝细胞生长因子-NK4腺病毒的制备及初步鉴定

目的制备表达人肝细胞生长因子（HGF）-NK4蛋白的复制缺陷型重组腺病毒。方法酶切pcDNA3-hNK4质粒获得NK4基因编码区序列并克隆至穿梭载体构建重组pAdTrack—CMV—NK4载体，线性化后与pAdEasy-1共转化BJ5183，通过同源重组得到重组pAd—NK4病毒载体。将重组腺病毒载体转染HEK293包装细胞制备重组腺病毒，用病毒悬液感染人肝癌细胞株HepG2。RT—PCR法检测感染肿瘤细胞中NK4mRNA表达。结果酶切鉴定得到阳性pAd—NK4重组腺病毒载体，该载体能有效转染HEK293细胞并在细胞内成功包装。在转染2d后能观察到绿色荧光蛋白（GFP）表达。制备的Ad—NK4在体外能有效感染HepG2细胞并获得NK4基因高水平表达。结论成功构建了NK4基因的重组腺病毒载体并制备重组腺病毒颗粒，为进一步研究NK4基因的功能及应用NK4进行基因治疗提供实验依据。     

重组腺病毒载体靶向性策略的研究进展

近年来,研究者致力于突破基因治疗的瓶颈,极大推动了基因治疗的发展。载体系统是基因治疗的基础,截止2013年7月,在"Gene Therapy Clinical Trial Worldwide"网站登记注册的基因治疗方案中,重组腺病毒载体占476项(23.5%)。在6个亚群(A-F)、50多种血清型的腺病毒载体中,5型腺病毒(adenovirus type 5,Ad5)可特异性识     

表达GFP基因的溶瘤腺病毒对大肠癌细胞的体外杀伤作用

目的： 研究在端粒酶启动子驱动下表达绿色荧光蛋白（GFP）及 5型腺病毒早期基因（E1A）基因的腺病毒Ad/hTERT-GFP-E1对大肠癌细胞的杀伤作用及其可能的机制。方法:将不同滴度Ad/hTERT-GFP-E1感染大肠癌及正常细胞,以巨细胞病毒（CMV）启动子驱动下表达GFP基因的腺病毒Ad/CMV-GFP作为载体对照,通过半数组织培养感染量（TCID50）法测定病毒滴度及体外复制能力,然后用MTT法及细胞克隆形成试验评价该病毒在体外对肿瘤细胞及正常细胞的杀伤作用,并对病毒感染后的细胞进行原位凋亡检测。结果:Ad/hTERT-GFP-E1能够在DLD1细胞内持续复制,GFP的表达能够对病毒的感染和复制起到监测作用;MTT结果显示该病毒对于结直肠肿瘤细胞有显著杀伤作用,而对于正常细胞没有明显的杀伤作用;对病毒感染后的DLD1进行细胞凋亡检测发现Ad/hTERT-GFP-E1所引起的凋亡率显著高于对照组(P＜0.01)。结论:溶瘤腺病毒Ad/hTERT-GFP-E1能够选择性地在肿瘤细胞内复制并杀伤肿瘤细胞,其机制与细胞凋亡的途径有关。     

CXCR4在哮喘小鼠肺组织中的表达及氯喹的干预作用

目的检测哮喘小鼠中趋化因子受体CXCR4的表达及其拮抗剂氯喹(CQ)的干预作用。方法将6~8周SPF级Balb/c小鼠随机分成对照组、哮喘组和氯喹干预组,哮喘组和氯喹干预组用鸡卵清蛋白(OVA)致敏和激发建立哮喘小鼠模型,干预组于激发前30 min给予CQ,连续3 d,对照组用PBS代替。最后1次激发24 h内小鼠肺功能仪检测小鼠气道高反应;最后1次激发48 h内检测支气管肺泡灌洗液(BALF)中细胞总数及细胞分类计数;HE染色光镜下观察肺组织的病理炎症改变,免疫组化染色检测肺组织CXCR4的表达;荧光定量PCR(Q-PCR)测定肺组织中CXCR4 mRNA的表达。结果哮喘组气道高反应及BALF中细胞总数明显高于对照组,干预组小鼠气道高反应及BALF中细胞总数与哮喘组相比显著降低;BALF中哮喘组嗜酸性粒细胞、中性粒细胞和单核细胞数较空白组显著增加,而氯喹干预组较哮喘组显著减低;HE染色结果显示哮喘组肺部炎症细胞浸润明显,干预组与哮喘组相比明显减轻,病理评分降低;免疫组化显示哮喘组CXCR4在气道上皮细胞呈强阳性表达,干预组表达呈弱阳性;哮喘组CXCR4 mRNA的表达较对照组升高,氯喹干预组较哮喘组CXCR4 mRNA表达量降低;CXCR4表达量与BALF细胞总数呈正相关。结论 CXCR4可能参与哮喘小鼠的气道炎症和气道高反应的发生,氯喹能改善哮喘小鼠的气道高反应和气道炎症可能与拮抗CXCR4相关,为氯喹治疗哮喘提供理论和实验依据,为开发治疗哮喘新药提供新的思路。     

5Aza-dc对癌细胞TIMP-3启动子去甲基化的作用

目的：观察5-氮杂-2'-脱氧胞苷（5-Aza-2'-deoxycytidine，5Aza-dc）对癌细胞TIMP-3启动子甲基化的影响。 方法： 用5Aza-dc处理TIMP-3启动子甲基化的H2M肝癌细胞和A431表皮癌细胞，用Transwell检测癌细胞的侵袭及运动能力，用Western blot 检测TIMP-3的蛋白表达，用RT-PCR检测TIMP-3 mRNA的表达，用甲基化特异性PCR检测TIMP-3基因启动子的甲基化。 结果： （1）5Aza-dc作用后的H2M和A431细胞的侵袭及运动能力降低；（2）5Aza-dc作用后的H2M和A431细胞的TIMP-3蛋白及mRNA表达量增加；（3）5Aza-dc作用后的H2M和A431细胞的TIMP-3启动子区未检测到甲基化。 结论： 5Aza-dc可以使肝癌和表皮癌细胞TIMP-3启动子区去甲基化，使TIMP-3得以重新表达，恢复其抑制肿瘤侵袭和移动的能力。     

SDF-1/CXCR4生物学轴与肿瘤关系的研究进展

趋化因子SDF-1,又称CXCL12,PBSF,与其特异性受体CXCR4广泛表达在多种组织和器官上,它们所构成的SDF-1/CXCR4 生物学轴在多种肿瘤的发生,发展以及转移中都发挥重要作用,其可能是通过MAPK,AKT通路发挥作用。对这一特殊生物学轴的研究可能为肿瘤防治找到新的突破口。     

重组人肝细胞生长因子-NK4腺病毒的制备及初步鉴定

目的 制备表达人肝细胞生长因子(HGF)-NK4蛋白的复制缺陷型重组腺病毒.方法 酶切pcDNA3-hNK4质粒获得NK4基因编码区序列并克隆至穿梭载体构建重组pAdTrack-CMV-NK4载体,线性化后与pAdEasy-1共转化BJ5183,通过同源重组得到重组pAd-NK4病毒载体.将重组腺病毒载体转染HEK293包装细胞制备重组腺病毒,用病毒悬液感染人肝癌细胞株HepG2.RT-PCR法检测感染肿瘤细胞中NK4 mRNA表达.结果 酶切鉴定得到阳性pAd-NK4重组腺病毒载体,该载体能有效转染HEK293细胞并在细胞内成功包装.在转染2d后能观察到绿色荧光蛋白(GFP)表达.制备的Ad- NK4在体外能有效感染HepG2细胞并获得NK4基因高水平表达.结论 成功构建了NK4基因的重组腺病毒载体并制备重组腺病毒颗粒,为进一步研究NK4基因的功能及应用NK4进行基因治疗提供实验依据.     

CXCR4与肿瘤的发生和发展

趋化因子受体-4（chemokinereceptor-4,CXCR4）属趋化因子家族,为G蛋白偶联的7次跨膜受体蛋白,基质细胞衍生因子-12是该受体的唯一配体。目前发现CXCR4在23种不同类型肿瘤中均有表达,与肿瘤细胞的增殖、侵袭、转移及预后密切相关,针对CXCR4靶向治疗可望成为肿瘤基因治疗研究的新热点。     

抗CD4及抗CXCR4抗体阻断HIV—1感染细胞作用的研究

为探讨抗ＣＤ４ 及抗ＣＸＣＲ４ 抗体在阻断Ｉ型人免疫缺陷病毒（ＨＩＶ－ １） 感染应用中的意义, 本文应用上述两种抗体分别与ＳｕｐＴ１ 细胞及人外周血单个核细胞（ＰＢＭＣ） 共培育, 以封闭ＨＩＶ－ １ 在上述细胞上的受体。然后, 以ＨＩＶ１ ＮＬ４３ 病毒株感染上述细胞, 通过测定感染细胞上清中ＨＩＶ－ １ 的Ｐ２４ 蛋白含量, 观察上述抗体对ＨＩＶ－ １ 感染细胞的阻断作用。结果显示, 无论是抗ＣＤ４ 或抗ＣＸＣＲ４ 的抗体单独应用或是两者联合应用, 均可明显地抑制ＨＩＶ－ １ 感染细胞的作用。该结果为今后开拓ＡＩＤＳ的抗体治疗提供了理论基础。     

Early infection and spread of a conditionally replicating adenovirus under conditions of plaque formation

Conditionally-replicating adenoviruses (CRAds) and other oncolytic viruses replicate selectively in tumor cells, presenting a potential cancer treatment approach. To optimize application of these viruses, understanding of early spread of these viruses in target cells is important. Here we used a recombinant adenovirus expressing enhanced jellyfish green fluorescent protein (EGFP) in place of the EIA and EIB genes (AdEGFPuci). Infection of susceptible cells (AD-293) under plaque formation conditions (MOI < < 1) on gridded culture dishes and daily monitoring allowed visualization of initially infected cells, as well as spread to neighboring cells. We determined key parameters of early infection, including the rate and efficiency of spread from the initially infected cell to other cells. It was noteworthy that a minority of initially infected cells ultimately resulted in plaques. The approaches elucidated here will be useful for determining early infection parameters for CRAds of therapeutic interest.     

内毒素对新生大鼠肺组织CXCR4表达影响的研究

目的研究内毒素作用于新生大鼠后,肺组织CXCR4表达的变化及可能作用。方法用内毒素诱发7日龄新生大鼠急性肺损伤(ALI),免疫组织化学法检测肺组织中CXCR4及TNF-α蛋白水平的表达情况。结果正常新生大鼠肺组织中CXCR4及TNF-α蛋白呈阴性或弱阳性表达,LPS作用后,CXCR4及TNF-α蛋白的表达随着损伤严重程度的增加而增强。结论 CXCR4可能在新生大鼠急性肺损伤的发病中发挥一定的作用。     

CXCR4阳性Lewis肺癌细胞耐药实验研究

目的 研究CXCR4阳性Lewis肺癌细胞(Lewis lung carcinoma,LLC)原发性耐药机制.方法 激光共聚焦检测小鼠移植瘤组织内CXCR4阳性LLC;以CXCR4作为磁珠分选细胞的表面标志,应用CCK-8法检测CXCR4阳性和阴性LLC对顺铂的敏感性,RT-PCR检测两者ABCG2、IGF1R mRNA表达情况.结果 小鼠移植瘤组织内散在分布胞膜呈红色荧光的CXCR4阳性LLC;CXCR4阳性与阴性LLC比较,具有更强的增顺铂抗拒性(P<0.05);CXCR4阳性LLC的ABCG2 mRNA表达(0.5240±0.0078)明显高于CXCR4阴性LLC(0.3870±0.0066) ,相差显著(P<0.01);CXCR4阳性LLC的 IGF1R mRNA表达(0.4209 ±0.0074)明显高于CXCR4阴性LLC(0.1848±0.0066),相差显著(P<0.01).结论 Lewis肺癌细胞中的CXCR4阳性亚群具有更强上调ABCG2、IGF1R表达的能力,具有顺铂抗拒性.     

CXCR4 and cancer

The chemokine receptor CXCR4 belongs to the large superfamily of G protein‐coupled receptors and has been identified to play a crucial role in a number of biological processes, including the trafficking and homeostasis of immune cells such as T lymphocytes. CXCR4 has also been found to be a prognostic marker in various types of cancer, including leukemia and breast cancer, and recent evidence has highlighted the role of CXCR4 in prostate cancer. Furthermore, CXCR4 expression is upregulated in cancer metastasis, leading to enhanced signaling. These observations suggest that CXCR4 is important for the progression of cancer. The CXCR4‐CXCL12 (stromal cell‐derived factor 1 (SDF‐1)) axis has additionally been identified to have a role in normal stem cell homing. Interestingly, cancer stem cells also express CXCR4, indicating that the CXCR4‐SDF‐1 axis may direct the trafficking and metastasis of these cells to organs that express high levels of SDF‐1, such as the lymph nodes, lungs, liver, and bone. This review focuses on the current knowledge of CXCR4 regulation and how deregulation of this protein may contribute to the progression of cancer.     

趋化因子及其受体CXCL12/CXCR4在人前列腺癌转移机制中的作用

目的 探讨趋化因子及其受体CXCL12/CXCR4在人前列腺癌转移机制中的作用.方法 免疫组织化学技术分析CXCL12/CXCR4蛋白在18例前列腺癌组织中的表达;免疫细胞化学技术分析CXCL12/CXCR4蛋白在人前列腺癌细胞株PC3、DU145和LNCap中的表达;迁移、侵袭试验分析外源性CXCL12对PC3、DU145和LNCap体外侵袭能力的调节作用.结果 18例人前列腺癌组织中,17例不同强度表达CXCR4蛋白,1例阴性表达,同时除1例标本弱表达CXCL12蛋白外,其余不表达CXCL12蛋白.3种前列腺癌细胞株均表达CXCR4蛋白,不表达CXCL12蛋白.外源性CXCLl2可明显促进PC3、DU145及LNCap的体外迁移、侵袭,以抗CXCL12或CXCR4抗体预处理PC3、LNCap细胞可以拮抗CXCL12对它们的促迁移、侵袭作用.结论 人前列腺癌组织表达CXCR4蛋白,CXCL12/CXCR4信号通路可能参与前列腺癌的侵袭、转移.     

The CXCR4/CXCL12 axis in endometrial cancer

Chemokines and their receptors seem to act as important regulators of the metastatic cascade. CXCL12 and its receptor CXCR4 were shown to be involved in human cancer progression. There is increasing evidences suggesting that the expression of CXCR4 in human cancers is correlated with poor patient prognosis and that CXCR4 neutralization can prevent metastases in vivo. Here we tested the role of the CXCR4/CXCL12 axis in a neoplasia with a reduced risk of metastatic progression, such as human endometrial cancer. CXCR4 and CXCL12 mRNA expression was measured in 41 endometrial cancers and in corresponding not affected tissues. The expression of CXCR4 was predominant in endometrial cancer (P = 0.035) whereas CXCL12 was overexpressed in normal mucosae (P = 0.002). CXCR4 expression (P = 0.035), but not CXCL12, was significantly related to cancer differentiation. Endometrial cancer cells (HEC1A) were able to generate diffuse metastases in peritoneum, lung and liver of CD-1 nude mice, but the simultaneous treatment with a neutralizing anti-CXCR4 monoclonal antibody dramatically reduced the number and the size of metastases in the animals. In conclusion, our data seem to indicate that the CXCR4-CXCL12 axis can play a role in the progression of endometrial carcinoma and that specific therapies with antagonists of chemokines receptors could be of help in the treatment of metastatic patients.     

Co-expression of uPAR and CXCR4 promotes tumor growth and metastasis in small cell lung cancer

Urokinase-type plasminogen activator receptor (uPAR) and C-X-C-chemokine receptor-4 (CXCR4) are considered as key molecules in invasion and metastasis of several cancers via extracellular matrix degeneration and assist tumor metastasis to specific sites by chemotaxis. However, the combined effect of uPAR and CXCR4 on small cell lung cancer (SCLC), the most aggressive type of lung cancer, is not clear. In this study, we detected the expression of uPAR and CXCR4 in SCLC tissue samples (n = 50) by immunohistochemistry. The tumors with high expression of both uPAR and CXCR4 (12/50) had larger size, higher lymph node (LN) metastasis and worse prognosis of patients than those with low expression of uPAR and CXCR4 (38/50) (P < 0.05). We further identified and isolated the both uPAR and CXCR4 positive expression subpopulation cells (uPAR⁺CXCR4⁺ cells) from the SCLC cell line H446 by flow cytometry. The uPAR⁺CXCR4⁺ cancer cells showed a higher invasive and migrating capacity in the transwell and wound healing assays compared with other subpopulation cells (P < 0.05). uPAR⁺CXCR4⁺ cells injected subcutaneously in nude mice markedly increased tumor growth and induced lung metastasis, while other subpopulation cells did not. In conclusion, these data suggest that uPAR and CXCR4 co-expression predicts worse prognosis of SCLC patients. uPAR⁺CXCR4⁺ cells promote the tumor growth and play a potential role in metastasis of SCLC.     

甲状腺癌组织中趋化因子4受体和趋化因子7受体的表达情况及临床病理意义

目的 研究甲状腺癌组织中趋化因子4受体(CXCR4)和趋化因子7受体(CXCR7)的表达情况及临床病理恿义.方法 选取2012年5月至2014年6月期间在本院进行外科手术的83例甲状腺癌患者及2014年45例甲状腺腺瘤患者,采用免疫组织化学染色方法检测甲状腺腺瘤及甲状腺癌中CXCR4和CXCR7的表达.结果 CXCR4和CXCR7在甲状腺癌中的表达阳性率均明显高于甲状腺腺瘤(P＜0.05).CXCR4和CXCR7在临床分期为Ⅲ～Ⅳ期的甲状腺癌患者中的表达阳性率均明显高于临床分期为Ⅰ～Ⅱ期者(P ＜0.05);CXCR7在有淋巴结转移的甲状腺癌患者中的表达阳性率明显高于无淋巴结转移者(P＜0.05),而CXCR4的表达阳性率与甲状腺癌患者有无淋巴结转移无关(P＞0.05);CXCR4和CXCR7在甲状腺癌组织中的表达阳性率与甲状腺癌患者性别无关(P＞0.05).在甲状腺癌患者中,CXCR4阳性表达和CXCR7阳性表达呈正相关(r =0.49,P＜0.01);在甲状腺腺瘤患者中,CXCR4阳性表达和CXCR7阳性表达不相关(r=0.14,P=0.21).结论 CXCR4和CXCR7参与了甲状腺癌的进展,并为临床上甲状腺癌诊断及靶向治疗提供理论基础.