反义核酸对人大肠癌CCL229细胞侵袭力的抑制作用

为观察反义寡核苷酸(asODN)对培养的高侵袭性人大肠癌CCL229细胞侵袭性的抑制作用,针对尿激酶型纤溶酶原激活物受体(re-ceptorofuPAR)mRNA的翻译起始区,合成了一段反义寡核苷酸(asODN),用脂质体将其导入培养的CCL229细胞中,通过逆转录-聚合酶链反应(RT-PCR)、流式细胞术、羊膜侵袭试验测定uPARmRNA水平、细胞表面uPAR抗原表达及细胞侵袭力的变化,并在扫描电镜下观察细胞的形态改变.结果为(1)RT-PCR检测asODNs组uPAR/β-actin比值为0.44±0.02,与对照组(0.81±0.01)相比,显著降低(P＜0.05);(2)流式细胞术检测asODNs组癌细胞表面与uPA结合的受体和总受体的平均荧光指数分别为0.20±0.07、0.59±0.09,与对照组(分别为0.72±0.12、2.21±0.36)比较,明显下降(P＜0.01,P＜0.05);(3)体外侵袭试验结果,asODN组在48h和72h后穿过羊膜的细胞数分别为12±2、20±3,与对照组(分别为25±4、44±5)相比,明显减少;(4)扫描电镜观察表明细胞经asODNs作用后,表面伪足和针状突起明显减少.以上各指标在随机序列寡核苷酸(rODN)组和对照组相比,无明显变化.CCL229细胞表面uPAR的表达与癌细胞的侵袭力密切相关,asODNs能有效抑制CCL229细胞uPAR基因的表达,从而抑制相关的蛋白质合成,降低其侵袭性,实验结果在癌症的基因治疗上将具有一定意义.     

特异性下调GRP94对人大肠癌细胞生物学特性的影响

为观察GBP94对培养的人大肠癌细胞系CCL229生物学特性的影响,将特异性裂解GBP94 mRNA翻译起始区的核酶,用脂质体介导的转染方法导入培养的CCL229细胞中。在确定获得稳定转染株后,我们检测了细胞生物学特性的变化。结果为：(1)转染GRP94核酶的细胞在A23187诱导16h后,细胞生长率显降低。(2)核酶表达细胞诱导后的聚集能力明显下降。(3)核酶表达细胞在A23187诱导后,停滞在G0／G1期的比例明显升高。结论为GBP94与应激的大肠癌细胞的生长和侵袭能力密切相关;特异性下调GBP94能改变人大肠癌细胞系CCL29的一些生物学特性。实验结果为深入研究GRP94与肿瘤细胞发生、发展和转移的关系奠定了基础,在癌症的基因治疗上将具有一定意义。     

维甲酸诱导的人大肠癌细胞凋亡

本研究应用光镜、电镜技术、DNA凝胶电泳、流式细胞术及末端脱氧核苷酰转移酶原位标记(TUNEL法),观察全反式维甲酸ATRA诱导的人大肠癌CCL229细胞凋亡特征。RA诱导CCL229细胞凋亡,光、电镜下观察到凋亡小体形成等典型的形态学改变,琼脂糖凝胶电泳上呈现特征性的DNA ladder,DNA直方图上显示亚二倍体峰。10-8mol/L-105mol/L范围内,RA诱导CCL229细胞凋亡表现出时间和剂量依赖性。     

乙酰肝素酶促进骨肉瘤细胞增殖和侵袭的体外研究

目的:探讨通过基因转染正向调节HPSE-1,体外对骨肉瘤细胞系恶性特质的影响.方法:转染HPSE-1基因至骨肉瘤细胞系MG-63,检测HPSE-1 mRNA和蛋白水平的表达,进一步应用MTT试验和Transwell侵袭试验观察稳定转染的细胞的增殖力和侵袭力的影响.结果:成功建立稳定转染HPSE-1基因的MG-63细胞系MG-63-HPSE,且该细胞系在mRNA和蛋白水平均发现HPSE-1表达增高,MTT和Transwell试验结果发现MG-63-HPSE细胞的增殖力和侵袭力均明显高于对照组.结论:基因转染后过表达HPSE-1的骨肉瘤细胞系体外表现出增强的增殖和侵袭活性.     

冠状病毒核衣壳蛋白与延伸因子-1α的相互作用

目的:分析SARS冠状病毒(SARS-CoV)核衣壳蛋白(N蛋白)和229E冠状病毒(HCoV-229E)核衣壳蛋白与细胞延伸因子(EF)-1α的相互作用、翻译抑制效应及与多核细胞形成的关系。方法:构建、表达及纯化SARS-N蛋白和229E-N蛋白的GST融合蛋白,用GST-pull down方法分析其与过表达的EF-1α及内源EF-1α之间的相互作用;构建和表达SARS-N蛋白和229E-N蛋白的GFP融合表达载体,转染293T细胞,通过共聚焦显微镜分析SARS-N蛋白和229E-N蛋白的亚细胞定位及多核细胞的形成;在293T细胞中过表达SARS-N蛋白或229E-N蛋白,通过Co-IP分析其与EF-1α的相互作用。分别在细胞内和体外翻译系统中分析二者抑制报告基因翻译的程度。结果:SARS-N蛋白和229E-N蛋白都定位于细胞质,并不像其他冠状病毒的N蛋白那样定位到细胞核;二者都能诱导形成多核细胞,但229E-N蛋白导致细胞形成多核细胞的时间要晚;二者都能与EF-1α相互作用并且共定位于细胞质,二者都能导致EF-1α形成多聚体;二者在细胞内及细胞外对报告基因都有抑制翻译效应。结论:SARS冠状病毒和229E冠状病毒的核衣壳蛋白均定位于细胞胞质,可与EF-1α相互作用,导致EF-1α形成多聚体,抑制报告基因翻译及导致细胞形成多核。     

趋化因子CCL2对人脐静脉内皮细胞ICAM-1 表达的影响

目的:探讨CC类趋化因子配体2(C-C motif ligand 2,CCL2)对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)中细胞间粘附分子-1(intercellular adhesion molecule-1,ICAM-1)表达的影响。方法:体外分离培养HUVECs细胞,将HUVECs铺至6孔板中,待细胞融合至80-90%时,将CCL2过表达载体[pc DNA3.1(+)-CCL2]及CCL2小分子干扰RNA(si-RNA)分别转染到HUVECs中,于转染后12 h、24 h和48 h收集细胞进行RNA及蛋白提取。荧光定量PCR方法检测HUVECs中CCL2及ICAM-1基因m RNA表达。Western blotting检测HUVECs中CCL2及ICAM-1蛋白表达。结果:(1)与pc DNA3.1(+)组相比较,pc DNA3.1(+)-CCL2组中CCL2基因m RNA和蛋白水平均显著升高;与si-Control组相比较,si-CCL2组中CCL2基因m RNA和蛋白表达均明显下降。(2)与对照组比较,pc DNA3.1(+)-CCL2组明显增加HUVECs中ICAM-1的m RNA及蛋白表达,而si-CCL2组显著抑制HUVECs中ICAM-1的m RNA及蛋白表达。结论:CCL2能增加HUVECs中ICAM-1基因m RNA和蛋白表达,为深入认识动脉粥样硬化的发病机制提供了理论依据。     

生半夏、南星水提物对人胃癌BGC823 细胞的侵袭力及 HIF-1αmRNA 蛋白表达的影响

目的:观察生半夏、南星中药水提物对缺氧环境中人胃癌细胞株BGC823细胞HIF-1α蛋白表达和侵袭力的影响。方法:运用CoCl(2氯化钴)诱导BCG823细胞缺氧,使得细胞中HIF-1α蛋白表达升高.实验组加入生半夏、南星水提物对细胞进行预处理,然后在进行缺氧诱导。通过甲基噻唑基四唑法(MTT)检测细胞活性,使用Transwell检测细胞侵袭能力变化,RT-PCR、Western blotting分别检测HIF-1α mRNA及蛋白含量及变化。结果:生半夏、南星水提物能抑制人胃癌BGC823细胞的增殖;生半夏、南星水提物均能抑制缺氧诱导胃癌细胞的侵袭力,并且能降低HIF-1α mRNA及蛋白表达。结论:生半夏、南星水提物可抑制人胃癌BGC823细胞的增殖,抑制人胃癌BGC823细胞侵袭力,可能通过降低HIF-1α蛋白表达有关。     

共表达IL-15和CCL19的EGFRvⅢ CAR-T细胞的构建和功能探究

本研究分析了共表达白细胞介素-15 (interleukin-15, IL-15)和趋化因子配体19 (C-C chemokine ligand 19, CCL19)的EGFRvⅢ CAR-T细胞的功能特性及其体外特异性杀伤效果，旨在优化CAR-T细胞多项功能，提高靶向EGFRvⅢ 的CAR-T细胞对胶质母细胞瘤(glioblastoma, GBM)的治疗效果。通过基因工程技术获得重组慢病毒质粒，转染293T细胞获得慢病毒并感染T细胞获得靶向EGFRvⅢ的第四代CAR-T细胞(EGFRvⅢ-IL-15-CCL19 CAR-T)。利用流式细胞仪、细胞计数仪、趋化小室、凋亡试剂盒等检测了第四代和第二代CAR-T细胞(EGFRvⅢ CAR-T)的CAR分子表达率、增殖、趋化能力、体外特异性杀伤能力及抗凋亡能力等。结果表明，与EGFRvⅢ CAR-T细胞相比，EGFRvⅢ-IL-15-CCL19 CAR-T细胞能成功分泌IL-15和CCL19，具有更强的体外增殖能力、趋化能力以及抗凋亡能力(P值均<0.05)，而体外特异性杀伤能力无显著差异。因此，靶向EGFRvⅢ且同时分泌IL-15和CCL19的CAR-T细胞有望提高胶质母细胞瘤的治疗效果，为临床试验提供一定的参考依据。     

生半夏、南星水提物对人胃癌BGC823细胞的侵袭力及HIF-lαmRNA蛋白表达的影响

摘要目的：观察生半夏、南星中药水提物对缺氧环境中人胃癌细胞株BGC823细胞HIF-lα蛋白表达和侵袭力的影响。方法：运用CoCl2（氯化钴）诱导BCG823细胞缺氧,使得细胞中HIF-1α蛋白表达升高．实验组加入生半夏、南星水提物对细胞进行预处理,然后在进行缺氧诱导。通过甲基噻唑基四唑法（MTT）检测细胞活性,使用Transwell检测细胞侵袭能力变化,RT-PCR、Weste-rn blotting分别检测HIF-lαmRNA及蛋白含量及变化。结果：生半夏、南星水提物能抑制人胃癌BGC823细胞的增殖;生半夏、南星水提物均能抑制缺氧诱导胃癌细胞的侵袭力,并且能降低HIF-1αmRNA及蛋白表达。结论：生半夏、南星水提物可抑制人胃癌BGC823细胞的增殖,抑制人胃癌BGC823细胞侵袭力,可能通过降低HIF-1α蛋白表达有关。     

人早孕滋养细胞分泌趋化因子CXCL16促进自身增殖和侵袭

本文探讨趋化因子CXCL16在细胞滋养细胞的分泌特征,以及其促进滋养细胞增殖和侵袭的生物学作用。采用原代培养人早孕滋养细胞,ELISA法检测不同种植密度培养12、24、36、48、60、 72、100h上清液中CXCL16分泌水平;[3H]TdR掺入法分析外源性CXCL16刺激后滋养细胞的增殖效应;matrigel侵袭试验分析外源性CXCL16处理后滋养细胞的侵袭性。结果表明原代培养的人早孕滋养细胞可持续分泌趋化因子CXCL16,在最初培养的48h分泌旺盛,培养至100h仍可见CX- CL16分泌;rhCXCL16浓度达100ng/ml时能够明显促进体外培养的滋养细胞增殖效应:rhCXCL16 浓度达10ng/ml时能明显促进滋养细胞的侵袭能力。实验证明人早孕滋养细胞分泌趋化因子CX- CL16,并以自分泌方式促进滋养细胞的增殖和侵袭,可能在胎盘形成和绒毛外滋养细胞的入侵中发挥重要作用。     

利用TNF-α诱导建立结肠癌细胞HCTll6体外侵袭模型

该研究利用TNF—α诱导建立肿瘤细胞体外侵袭模型,为进一步的研究提供基础。利用20ng／mLTNF-α刺激结肠癌细胞HCTll67d后,使用流式细胞术检测NHCTll6细胞的CCR7和CXCR4受体表达量的变化,并利用Transwell小室检测TNF—α刺激对CCL21、SDF－1介导的细胞迁移与侵袭能力的影响。实验结果显示,20ng／mLTNF－α刺激7d后的HCTll6细胞的CCR7和CXCR4表达量均显著增加,侵袭能力也增强,且使CCL21、SDF—1介导的细胞迁移与侵袭能力显著增强。结果说明了本实验利用TNF—α诱导HCTl16细胞,成功建立了HCTl16的体外侵袭模型,为接下来的研究提供了细胞模型基础,也为进一步的药物筛选提供了基础。     

Interferon treatment reduced adherence, invasiveness and intracellular multiplication of Shigella flexneri in coxsackie B1 virus-infected cells.

The effect of interferon treatment on interaction of Shigella flexneri with in vitro cultured cells was investigated. Pretreatment of HEp-2 cells with human interferons had no effect on the susceptibility of cells to S. flexneri, measured by invasiveness and adhesiveness. Human leukocyte interferon and human recombinant interferon-alpha-A reduced adhesiveness, intracellular multiplication and invasiveness of S. flexneri in HEp-2 cells preinfected with coxsackie B1 virus. Also non-receptor mediated-phagocytosis was reduced by interferon treatment in virus infected cells. The interferon effects were dependent on continuous protein synthesis, because they were not expressed when cycloheximide or abrin was added to the virus infected cell cultures. No effect of interferon was detected on intracellular content of Na+ or K+, Na(+)-K+ activated ATPase activity or cytoplasma membrane polarity, in virus infected or control cell cultures. The interferon effect on bacterial invasiveness seems to be dependent on an interferon receptor interaction on cytoplasma membrane level because directly microinjected interferon showed no effect.     

Ethanol treatment inhibits mesoderm cell spreading in the gastrulating chick embryo

Gastrulating chick embryos in culture were treated with ethanol solutions, following which the mesoderm cells migrating from the primitive streak were examined by scanning electron microscopy. Morphometric analysis of cell shape showed that mesoderm cells from treated embryos were significantly more rounded and therefore less well spread than controls, and showed fewer filopodial contacts with the overlying basement membrane. This result was only obtainable for cells leading the migration from the primitive streak, since the following cells in the mesodermal mass apparently did not show this difference. The ethanol concentration required to obtain a reliable effect was 5%, while lower concentrations produced highly variable results. The mesoderm cells were also examined for their in vitro responsiveness to ethanol by investigating their adhesiveness and cytoskeleton. No effect was observed on cell-glass adhesion as judged by interference reflection microscopy using up to 1% ethanol. This concentration did, however, disrupt the actin cytoskeleton of cultured cells when stained with NBD-phallacidin, but lower concentrations were ineffective. It is concluded that ethanol treatment of cultured embryos has a significant effect on the substratum relationships of some migrating mesoderm cells.     

Angiogenic activity of human CC chemokine CCL15 in vitro and in vivo

CCL15 is a novel human CC chemokine and exerts its biological activities on immune cells through CCR1 and CCR3. Because a number of chemokines induce angiogenesis and endothelial cells express CCR1 and CCR3, we investigated the angiogenic activity of CCL15. Both CCL15(1-92) and N-terminal truncated CCL15(25-92) stimulate the chemotactic endothelial cell migration and differentiation, but CCL15(25-92) is at least 100-fold more potent than CCL15(1-92). Treatment with pertussis toxin (PTX), with anti-CCR1, or with anti-CCR3 antibody inhibits the CCL15(25-92)-induced endothelial cell migration. CCL15(25-92) also stimulates sprouting of vessels from aortic rings and mediates angiogenesis in the chick chorioallantoic membrane assay. Our findings demonstrate that CCL15(25-92) has in vitro and in vivo angiogenic activity, and suggest roles of the chemokine in angiogenesis.     

Preferential migration of regulatory T cells mediated by gliomasecreted chemokines can be blocked with chemotherapy

Despite the immunogenicity of glioblastoma multiforme (GBM), immune-mediated eradication of these tumors remains deficient. Regulatory T cells (Tregs) in the blood and within the tumor microenvironment of GBM patients are known to contribute to their dismal immune responses. Here, we determined which chemokine secreted by gliomas can preferentially induce Treg recruitment and migration. In the malignant human glioma cell lines D-54, U-87, U-251, and LN-229, the chemokines CCL22 and CCL2 were detected by intracellular cytokine analysis. Furthermore, tumor cells from eight patients with GBM had a similar chemokine expression profile. However, only CCL2 was detected by enzyme-linked immunosorbent assay, indicating that CCL2 may be the principal chemokine for Treg migration in GBM patients. Interestingly, the Tregs from GBM patients had significantly higher expression levels of the CCL2 receptor CCR4 than did Tregs from healthy controls. Glioma supernatants and the recombinant human chemokines CCL2 and CCL22 induced Treg migration and were blocked by antibodies to the chemokine receptors. Production of CCL2 by glioma cells could also be mitigated by the chemotherapeutic agents temozolomide and carmustine [3-bis (2-chloroethyl)-1-nitrosourea]. Our results indicate that gliomas augment immunosuppression by selective chemokine-mediated recruitment of Tregs into the tumor microenvironment and that modulating this interaction with chemotherapy could facilitate the development of novel immunotherapeutics to malignant gliomas. Justin T. Jordan and Wei Sun are contributed equally to this work. An erratum to this article can be found at     

Human CC chemokine CCL23, a ligand for CCR1, induces endothelial cell migration and promotes angiogenesis

A number of chemokines induce angiogenesis and endothelial cells express several chemokine receptors. To date, only a limited number of CC chemokines for CCR1 have been reported to induce angiogenic responses. We investigated the ability of CCL23 (also known as MPIF-1, MIP-3, or CKbeta8) to promote angiogenesis, which induces chemotaxis of immune cells through CCR1. CCL23 promoted the chemotactic migration and differentiation of endothelial cells, and neovascularization in the chick chorioallantoic membrane. An N-terminal truncated form of CCL23 was at least 100-fold more potent than its intact form and was comparable to that of FGF in the angiogenic activities. Treatment with either pertussis toxin or anti-CCR1 antibody completely inhibited the CCL23-induced endothelial cell migration, indicating that endothelial cell migration was mediated through CCR1. CCL23 didn't promote the migration of HT1080 human fibrosarcoma cells that did not express CCR1. Our results suggest a role of CCL23 in angiogenesis in vitro as well as in vivo.     

CCL229细胞经诱导后蛋白激酶C及抑制剂活性的变化

检测以维甲酸(RA), 1, 25－二羟基维生素D₃(1,25(OH)₂VD₃)诱导2d、佛波酯（PMA）诱导6h的人大肠癌细胞CCL₂₂₉的蛋白激酶C（PKC）及其抑制剂活性．结果显示：诱导后PKC总活性升高(P<0.05); RA, 1, 25(OH)₂VD诱导引起细胞质PKC活性增加,PMA诱导后细胞膜PKC比率（细胞膜活性／总活性）显著升高(P<0.01);诱导后PKC抑制剂活性均降低,其中1, 25 (OH)₂VD₃组与对照组有显著差异(P<0.05);提示PMA引起PKC从细胞质向细胞膜转移,不同药物诱导后PKC及其抑制剂活性出现不同的相对均衡关系．