Fra-1对大鼠C6胶质瘤细胞侵袭转移能力的影响

目的：观察Fra-1对C6胶质瘤侵袭转移能力的影响。方法：以大鼠C6胶质瘤细胞为研究对象,Fra-1siRNA通过脂质体转染C6,realtimeRT-PCR和westernblot法检测C6细胞中Fra-1的表达、Elisa法检测细胞培养上清中MMP一9的含量,Transwell小室观察C6细胞的转移侵袭能力。结果：干扰Fra-1后能降低C6细胞中Fra-1的表达,显著降低C6细胞中MMP-9的含量,降低C6细胞的转移侵袭能力。结论：干扰Fra-1能抑制C6细胞的转移侵袭。     

ID1对大鼠C6细胞侵袭转移能力的影响

目的:观察ID1对大鼠胶质瘤细胞(C6细胞)侵袭转移能力的影响。方法:以C6细胞为研究对象,ID1siRNA通过脂质体转染C6细胞,westernblot检测C6细胞中ID1、P-NF-КBp65蛋白的表达、Elisa法检测细胞培养上清MMP-9的含量,Transwell小室观察成的转移侵袭能力。结果:干扰ID1后能显著降低C6细胞中P-NF-КBp65蛋白、细胞培养上清中MMP-9的含量(P<0.01),降低C6细胞的转移侵袭能力(P<0.01)。结论:干扰ID1能抑制C6细胞的转移侵袭,其机制可能与抑制C6细胞中P-NF-КBp65蛋白、MMP-9的表达有关。     

ID1对大鼠C6细胞侵袭转移能力的影响

目的：观察IDl对大鼠胶质瘤细胞（c6细胞）侵袭转移能力的影响。方法：以C6细胞为研究对象,ID1 siRNA通过脂质体转染c6细胞,western blot检测C6细胞中ID1、P-NF—KBp65蛋白的表达、Elisa法检测细胞培养上清MMP．9的含量,Transwell小室观察成的转移侵袭能力。结果：干扰ID1后能显著降低C6细胞中P-NF-KBp65蛋白、细胞培养上清中MMP-9的含量（P〈0．01）,降低C6细胞的转移侵袭能力（P〈0．01）。结论：干扰ID1能抑制C6细胞的转移侵袭,其机制可能与抑制C6细胞中P-NF-K Bp65蛋白、MMP-9的表达有关。     

SFRP4抑表达对HT-29细胞侵袭能力的影响

目的:观察SFRP4对HT29细胞侵袭转移能力的影响。方法:以HT29细胞为研究对象,SFRP4 siRNA通过脂质体转染HT29细胞,western blot检测HT29细胞中Wnt、细胞核β-Catenin蛋白的表达、elisa法检测细胞培养上清MMP-2、MMP-9的含量,Transwell小室观察成的转移侵袭能力。结果:干扰SFRP4后能显著降低HT29细胞中Wnt、细胞核中β-Catenin蛋白的表达、细胞培养上清MMP-2、MMP-9的含量,(P<0.01),降低HT29细胞的转移侵袭能力(P<0.01)。结论:抑制SFRP4表达能抑制HT29细胞的转移侵袭,其机制可能与抑制HT29细胞Wnt信号通路有关。     

SFRP4抑表达对HT-29细胞侵袭能力的影响

目的：观察SFRP4对HT29细胞侵袭转移能力的影响。方法：以HT29细胞为研究对象,SFRP4siRNA通过脂质体转染HT29细胞,westernblot检测HT29细胞中wnt、细胞核β—Catenin蛋白的表达、elisa法检测细胞培养上清MMP-2、MMP．9的含量,Transwell小室观察成的转移侵袭能力。结果：干扰SFRP4后能显著降低HT29细胞中Wnt、细胞核中B—Catenin蛋白的表达、细胞培养上清MMP-2、MMP-9的含量,（P〈0．01）,降低HT29细胞的转移侵袭能力（P〈0．01）。结论：抑制SFRP4表达能抑制HT29细胞的转移侵袭,其机制可能与抑制HT29细胞wnt信号通路有关。     

人参皂苷Rh2对大鼠C6胶质瘤细胞Siah-1、Synaptophysin、MMP9及VEGF表达的影响

摘要 目的：探讨人参皂苷Rh2对大鼠C6胶质瘤细胞Siah-1、突触素（Synaptophysin）、基质金属蛋白酶-9（MMP-9）血管内皮生长因子（VEGF）表达的影响。方法：将大鼠C6胶质瘤细胞分为对照组、人参皂苷Rh2低剂量组（16 μg/mL）、人参皂苷Rh2中剂量组（32 μg/mL）、人参皂苷Rh2高剂量组（48 μg/mL），CCK-8法和平板克隆实验检测细胞增殖；流式细胞术检测细胞凋亡；Transwell检测细胞侵袭；实时荧光定量-聚合酶链式反应（qRT-PCR）检测C6胶质瘤细胞中VEGF、Siah-1、Synaptophysin、MMP-9 mRNA表达；蛋白质印迹法（Western blot）检测C6胶质瘤细胞中VEGF、Siah-1、Synaptophysin、MMP-9蛋白表达。结果：与对照组比较，人参皂苷Rh2低剂量组、人参皂苷Rh2中剂量组、人参皂苷Rh2高剂量组大鼠C6胶质瘤细胞OD₄₅₀值（24 h、48 h）、克隆形成率、细胞侵袭数、VEGF、Synaptophysin、MMP-9 mRNA及蛋白表达降低，细胞凋亡率、Siah-1 mRNA及蛋白表达升高，且呈剂量依赖性（P<0.05）。结论：人参皂苷Rh2可能通过上调Siah-1，下调VEGF、Synaptophysin、MMP-9表达来抑制大鼠C6胶质瘤细胞增殖与侵袭，促进细胞凋亡。     

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.     

HMGB1 siRNA 对HT-29 细胞侵袭转移能力的影响

目的:观察干扰HMGB1表达对HT-29细胞侵袭转移能力的影响。方法:HMGB1 siRNA通过脂质体转染HT-29细胞,western blot和实时定量RT-PCR检测HT-29细胞中HMGB1蛋白和mRNA的表达,Transwell小室观察HT-29的转移侵袭能力。结果:干扰HMGB1后HMGB1蛋白和mRNA的表达均减少,HT-29的转移侵袭能力下降。结论:HMGB1能促进HT-29的转移侵袭能力,干扰其表达可抑制HT-29的转移侵袭。     

自分泌运动因子AMF对人胶质母细胞瘤U251细胞迁移、侵袭的影响及相关机制研究

为了探讨自分泌运动因子(autocrine motility factor,AMF)对人胶质母细胞瘤U251细胞迁移、侵袭影响及其相关分子机制,该实验采用了RT-PCR及免疫印迹法检测RNA干扰AMF后U251细胞中AMF的表达变化;细胞划痕实验、Transwell实验分别观察了AMF干扰前后U251细胞迁移、侵袭能力的变化;免疫印记检测AMF干扰前后细胞中总Akt、p-Akt、Sox2、基质金属蛋白酶-2(matrix metalloprotein-2,MMP-2)及MMP-9蛋白水平的变化。研究结果表明,AMF成功干扰后U251细胞的迁移和侵袭能力受到抑制,p-Akt、Sox2、MMP-2和MMP-9蛋白表达水平降低。该研究表明,AMF敲低可以通过下调PI3K/Akt信号通路活性及Sox2、MMP-2和MMP-9蛋白水平,抑制人胶质母细胞瘤U251细胞迁移和侵袭。     

地高辛通过下调AEG-1表达抑制人胃癌MKN45细胞迁移和侵袭

本研究旨在观察地高辛对人胃癌MKN45细胞迁移和侵袭能力的影响,并探讨其分子机制。取人胃癌MKN45细胞作为研究对象,采用Transwell法检测细胞迁移和侵袭能力,通过脂质体转染法将星形细胞上调基因-1(astrocyte elevated gene-1,AEG-1)sh RNA干扰质粒转染MKN45细胞以构建低表达AEG-1的细胞株,利用Western blot法检测基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)、E-钙粘蛋白(E-cadherin)和AEG-1等蛋白的表达变化。结果显示,地高辛可使MKN45细胞迁移率和侵袭率均显著下降(P0.05),下调MMP-9和AEG-1蛋白表达水平(P0.05)以及上调E-cadherin蛋白表达水平(P0.05),上述作用均具有剂量依赖性。经sh RNA干扰AEG-1基因表达后,MKN45细胞中AEG-1蛋白表达水平显著下降(P0.05);同时AEG-1干扰组MKN45细胞中E-cadherin蛋白表达水平显著升高(P0.05),细胞迁移率、侵袭率和MMP-9蛋白表达水平均显著下降(P0.05)。上述结果提示,地高辛可在体外剂量依赖性地抑制人胃癌MKN45细胞迁移和侵袭,这可能与地高辛抑制AEG-1蛋白表达,继而下调MMP-9蛋白表达和上调E-cadherin蛋白表达有关。     

HMGB1 siRNA对HT-29细胞侵袭转移能力的影响

目的：观察干扰HMGBl表达对HT-29细胞侵袭转移能力的影响。方法：HMGB1siRNA通过脂质体转染HT-29细胞,westernblot和实时定量RT—PCR检测HT-29细胞中HMGB1蛋白和mRNA的表达,Transwell小室观察HT-29的转移侵袭能力。结果：干扰HMGBl后HMGB1蛋白和mRNA的表达均减少,HT-29的转移侵袭能力下降。结论：HMGB1能促进HT-29的转移侵袭能力,干扰其表达可抑制HT-29的转移侵袭。     

Rhinovirus-induced MMP-9 expression is dependent on Fra-1, which is modulated by formoterol and dexamethasone

Matrix metalloproteinase-9 is implicated in airway inflammation and airway remodeling in asthma. We have previously confirmed that human rhinovirus-16 (HRV-16) infection increases MMP-9 expression both in vivo and in vitro. However, the role of the AP-1 sites within the MMP-9 promoter and the effect of commonly used asthma pharmacotherapies in modulating human rhinovirus (HRV)-induced MMP-9 production have not yet been elucidated. Experiments were performed in vitro in the human bronchial epithelial (HBE) cell line BEAS-2B and in primary HBE cells obtained from non-transplanted lungs. Using site-directed mutagenesis approaches, AP-1 sites were found to be necessary for HRV-induced MMP-9 promoter drive. EMSAs and supershift assays identified complexes consisting of Fos-related Ag-1 (Fra-1) in addition to other AP-1 subunits. Small interfering RNA approaches indicated that Fra-1 was induced upon HRV-16 infection in BEAS-2B cells and was necessary for MMP-9 expression in both BEAS-2B and primary HBE cells. Inhibition of MEK1/2 activity using PD98059 and U0126 reduced Fra-1 expression, DNA binding, MMP-9 promoter drive, and MMP-9 protein production. The long-acting β(2)-agonist formoterol and the glucocorticoid dexamethasone significantly reduced HRV-induced ERK phosphorylation, Fra-1, and MMP-9 expression in BEAS-2B cells. These data indicate that HRV-induced activation of the MEK/ERK MAPK pathway and Fra-1 expression are necessary for the upregulation of MMP-9 and can be modulated by two distinct but commonly used asthma pharmacotherapies. Together, these results offer insights into the mechanisms by which long-acting β(2)-agonists and glucocorticoids might reduce HRV-related asthma exacerbations.     

Phosphorylated c-Jun and Fra-1 induce matrix metalloproteinase-1 and thereby regulate invasion activity of 143B osteosarcoma cells

Osteosarcoma is the most common primary malignancy of bone and patients often develop pulmonary metastases. Despite the advances in surgical and medical management, the mechanisms underlying human osteosarcoma progression and metastasis remain to be elucidated. Gene expression profiles were compared by the cDNA microarray technique between two different human osteosarcoma sublines, MNNG/HOS and 143B, which differ greatly in spontaneous pulmonary metastatic potential. Here we report an enhanced expression of matrix metalloproteinase (MMP)-1 in the highly metastatic human osteosarcoma cell line 143B. Moreover, the in vitro invasion activity of 143B cells was MMP-1-dependent. The activator protein (AP)-1 binding site in the MMP-1 gene promoter was required for the constitutive expression of MMP-1 in 143B cells. Two AP-1 components, c-Jun and Fra-1, were phosphorylated, and bound to the AP-1 binding site of the MMP-1 promoter in 143B cells. Activated c-Jun and Fra-1 were essential for MMP-1 gene expression in 143B cells. Mitogen-activated protein kinase pathways including the c-Jun NH₂-terminal kinase and the extracellular signal-regulated kinase activate c-Jun and Fra-1 and thereby regulate c-Jun/Fra-1 mediated events, establishing the mitogen-activated protein kinase/AP-1/MMP-1 axis as important in 143B cells. These data suggest that MMP-1 plays a central role in osteosarcoma invasion. Accordingly, MMP-1 might be a biomarker and therapeutic target for invasive osteosarcomas and pulmonary metastases.     

可溶性endoglin减弱人早孕细胞滋养细胞的浸润功能

本研究旨在探讨可溶性endoglin(soluble endoglin,sEng)对人早孕细胞滋养细胞活力和浸润功能的影响。采用胰蛋白酶-DNase消化法培养人早孕期(孕6～8周)细胞滋养细胞,传代后待细胞长满至70%～80%,分别加入没有添加(对照组)和添加sEng(10μg/L)的细胞培养液培养24h;Transwell技术检测细胞滋养细胞的浸润功能;RT-PCR法检测细胞滋养细胞基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)、基质金属蛋白酶9(MMP-9)mRNA的表达;Western blot方法分别检测细胞滋养细胞MMP-2、MMP-9蛋白的表达。结果显示,sEng组细胞滋养细胞浸润能力低于对照组。与对照组比较,sEng组细胞滋养细胞MMP-2和MMP-9 mRNA及蛋白的表达明显下降(P<0.05)。以上结果提示,sEng可能通过调节人早孕细胞滋养细胞中MMP-2、MMP-9的表达而影响细胞的浸润能力,从而参与子痫前期的发生。