凝血因子Ⅶa促进SW620细胞增殖与迁移的机制探讨

目的 体外探讨凝血因子Ⅶa促进结肠癌细胞株SW620增殖与迁移的作用机制.方法 采用蛋白酶激活受体2激动剂(PAR2-AP)、凝血因子Ⅶa等处理SW620细胞,以实时定量PCR检测SW620细胞中白细胞介素8(IL-8)、组织因子(TF)及半胱氨酸蛋白酶7(caspase-7)mRNA的表达水平;以酶联免疫吸附试验(ELISA)检测细胞上清IL-8蛋白的含量;以Xa生成法检测细胞TF活性;以Western blot法检测细胞磷酸化p38丝裂原活化蛋白激酶(p-p38 MAPK)水平.结果 PAR2-AP、凝血因子Ⅶa能够增加SW620细胞中IL-8基因和蛋白的表达,上调TF mRNA水平及活性,下调caspase-7基因表达和p-p38 MAPK水平,单克隆抗TF及抗PAR2抗体均可抑制凝血因子Ⅶa的作用.结论 凝血因子Ⅶa与细胞表面TF形成复合物,通过活化PAR2,上调结肠癌细胞株SW620中IL-8、TF的表达,下调细胞caspase-7表达,从而促进细胞增殖与迁移能力,p38 IVIAPK在此过程中起负性调节作用.     

核因子κB在凝血因子Ⅶa促进结肠癌SW620细胞增殖迁移中的作用

目的 探讨核因子κB(NF-κB)在促进结肠癌SW620细胞增殖迁移过程中的作用及其可能的机制。方法 以凝血因子Ⅶa、NF-κB抑制剂二硫代氨基甲酸吡咯烷(PDTC)等处理结肠癌细胞株SW620,采用Western blot法检测细胞核NF-κB(p65)、细胞浆NF-κB抑制蛋白（IκB-o）和凋亡蛋白半胱氨酸蛋白酶7（caspase-7）的蛋白表达变化;采用流式细胞术检测SW620细胞的细胞周期变化;采用Transwell法测定SW620细胞的迁移能力;采用实时定量聚合酶链反应(PCR)检测白细胞介素8( IL-8)和组织因子(TF) mRNA的表达水平。结果凝血因子Ⅶa能够显著下调细胞浆中IκB-o的表达水平,并使细胞核内NF-κB的表达水平升高,单克隆抗TF和抗蛋白酶激活受体2(PAR2)抗体能够抑制凝血因子Ⅶa的这一作用。PDTC能够明显干预凝血因子Ⅶa对SW620细胞增殖、迁移的促进作用。PDTC能够明显干预凝血因子Ⅶa对SW620细胞中TF、IL-8 mRNA表达的促进作用和对caspase-7蛋白表达的下调作用。结论 凝血因子Ⅶa与细胞表面TF结合形成TF/Ⅶa复合物,活化受体PAR2,经NF-κB通路上调IL-8、下调caspase-7的表达,促进SW620细胞的增殖与迁移。TF/Ⅶa/PAR2/NF-κB通路还可进一步上调TF的表达,从而形成TF/Ⅶa/PAR2/NF-κB/TF正反馈通路。     

Investigation of the mechanisms of coagulation factor Ⅶa-induced colon cancer 8W620 cell proliferation and migration

Objective To investigate the mechanisms that coagulation factor Ⅶa promotes proliferation and migration of a colon cancer cell line (SW620 cells) in vitro. Methods The expression of interleukin 8 (IL-8), tissue factor (TF), caspase-7 and p-p38 MAPK in SW620 cells treated with factor Ⅶa or protease activated receptor 2 aganist (PAR2-AP) was measured by ELISA, Western-blotting and QT-PCR. Results Factor Ⅶa and PAR2-AP induced IL-8 expression at both mRNA and protein levels, up-regulated TF mRNA expression and TF activity, but down-regulated caspese-7 mRNA and p-p38 MAPK levels in SW620 cells. The effects of factor Ⅶa were not only blocked by anti-TF but also by anti-PAR2 antibodies. Conclusion Factor Ⅶa binds to TF on cell surface, forming a complex which activates PAR2, then provoking IL-8 and TF expression, and suppresses caepase-7 expression, thus promotes the tumor cell proliferation and migration, p38 MAPK may negatively regulate this process.     

Investigation of the mechanisms of coagulation factor Ⅶa-induced colon cancer 8W620 cell proliferation and migration

Objective To investigate the mechanisms that coagulation factor Ⅶa promotes proliferation and migration of a colon cancer cell line (SW620 cells) in vitro. Methods The expression of interleukin 8 (IL-8), tissue factor (TF), caspase-7 and p-p38 MAPK in SW620 cells treated with factor Ⅶa or protease activated receptor 2 aganist (PAR2-AP) was measured by ELISA, Western-blotting and QT-PCR. Results Factor Ⅶa and PAR2-AP induced IL-8 expression at both mRNA and protein levels, up-regulated TF mRNA expression and TF activity, but down-regulated caspese-7 mRNA and p-p38 MAPK levels in SW620 cells. The effects of factor Ⅶa were not only blocked by anti-TF but also by anti-PAR2 antibodies. Conclusion Factor Ⅶa binds to TF on cell surface, forming a complex which activates PAR2, then provoking IL-8 and TF expression, and suppresses caepase-7 expression, thus promotes the tumor cell proliferation and migration, p38 MAPK may negatively regulate this process.     

Investigation of the mechanisms of coagulation factor Ⅶa-induced colon cancer 8W620 cell proliferation and migration

Objective To investigate the mechanisms that coagulation factor Ⅶa promotes proliferation and migration of a colon cancer cell line (SW620 cells) in vitro. Methods The expression of interleukin 8 (IL-8), tissue factor (TF), caspase-7 and p-p38 MAPK in SW620 cells treated with factor Ⅶa or protease activated receptor 2 aganist (PAR2-AP) was measured by ELISA, Western-blotting and QT-PCR. Results Factor Ⅶa and PAR2-AP induced IL-8 expression at both mRNA and protein levels, up-regulated TF mRNA expression and TF activity, but down-regulated caspese-7 mRNA and p-p38 MAPK levels in SW620 cells. The effects of factor Ⅶa were not only blocked by anti-TF but also by anti-PAR2 antibodies. Conclusion Factor Ⅶa binds to TF on cell surface, forming a complex which activates PAR2, then provoking IL-8 and TF expression, and suppresses caepase-7 expression, thus promotes the tumor cell proliferation and migration, p38 MAPK may negatively regulate this process.     

Investigation of the mechanisms of coagulation factor Ⅶa-induced colon cancer 8W620 cell proliferation and migration

Objective To investigate the mechanisms that coagulation factor Ⅶa promotes proliferation and migration of a colon cancer cell line (SW620 cells) in vitro. Methods The expression of interleukin 8 (IL-8), tissue factor (TF), caspase-7 and p-p38 MAPK in SW620 cells treated with factor Ⅶa or protease activated receptor 2 aganist (PAR2-AP) was measured by ELISA, Western-blotting and QT-PCR. Results Factor Ⅶa and PAR2-AP induced IL-8 expression at both mRNA and protein levels, up-regulated TF mRNA expression and TF activity, but down-regulated caspese-7 mRNA and p-p38 MAPK levels in SW620 cells. The effects of factor Ⅶa were not only blocked by anti-TF but also by anti-PAR2 antibodies. Conclusion Factor Ⅶa binds to TF on cell surface, forming a complex which activates PAR2, then provoking IL-8 and TF expression, and suppresses caepase-7 expression, thus promotes the tumor cell proliferation and migration, p38 MAPK may negatively regulate this process.     

Investigation of the mechanisms of coagulation factor Ⅶa-induced colon cancer 8W620 cell proliferation and migration

Objective To investigate the mechanisms that coagulation factor Ⅶa promotes proliferation and migration of a colon cancer cell line (SW620 cells) in vitro. Methods The expression of interleukin 8 (IL-8), tissue factor (TF), caspase-7 and p-p38 MAPK in SW620 cells treated with factor Ⅶa or protease activated receptor 2 aganist (PAR2-AP) was measured by ELISA, Western-blotting and QT-PCR. Results Factor Ⅶa and PAR2-AP induced IL-8 expression at both mRNA and protein levels, up-regulated TF mRNA expression and TF activity, but down-regulated caspese-7 mRNA and p-p38 MAPK levels in SW620 cells. The effects of factor Ⅶa were not only blocked by anti-TF but also by anti-PAR2 antibodies. Conclusion Factor Ⅶa binds to TF on cell surface, forming a complex which activates PAR2, then provoking IL-8 and TF expression, and suppresses caepase-7 expression, thus promotes the tumor cell proliferation and migration, p38 MAPK may negatively regulate this process.     

Investigation of the mechanisms of coagulation factor Ⅶa-induced colon cancer 8W620 cell proliferation and migration

Objective To investigate the mechanisms that coagulation factor Ⅶa promotes proliferation and migration of a colon cancer cell line (SW620 cells) in vitro. Methods The expression of interleukin 8 (IL-8), tissue factor (TF), caspase-7 and p-p38 MAPK in SW620 cells treated with factor Ⅶa or protease activated receptor 2 aganist (PAR2-AP) was measured by ELISA, Western-blotting and QT-PCR. Results Factor Ⅶa and PAR2-AP induced IL-8 expression at both mRNA and protein levels, up-regulated TF mRNA expression and TF activity, but down-regulated caspese-7 mRNA and p-p38 MAPK levels in SW620 cells. The effects of factor Ⅶa were not only blocked by anti-TF but also by anti-PAR2 antibodies. Conclusion Factor Ⅶa binds to TF on cell surface, forming a complex which activates PAR2, then provoking IL-8 and TF expression, and suppresses caepase-7 expression, thus promotes the tumor cell proliferation and migration, p38 MAPK may negatively regulate this process.     

Investigation of the mechanisms of coagulation factor Ⅶa-induced colon cancer 8W620 cell proliferation and migration

Objective To investigate the mechanisms that coagulation factor Ⅶa promotes proliferation and migration of a colon cancer cell line (SW620 cells) in vitro. Methods The expression of interleukin 8 (IL-8), tissue factor (TF), caspase-7 and p-p38 MAPK in SW620 cells treated with factor Ⅶa or protease activated receptor 2 aganist (PAR2-AP) was measured by ELISA, Western-blotting and QT-PCR. Results Factor Ⅶa and PAR2-AP induced IL-8 expression at both mRNA and protein levels, up-regulated TF mRNA expression and TF activity, but down-regulated caspese-7 mRNA and p-p38 MAPK levels in SW620 cells. The effects of factor Ⅶa were not only blocked by anti-TF but also by anti-PAR2 antibodies. Conclusion Factor Ⅶa binds to TF on cell surface, forming a complex which activates PAR2, then provoking IL-8 and TF expression, and suppresses caepase-7 expression, thus promotes the tumor cell proliferation and migration, p38 MAPK may negatively regulate this process.     

Investigation of the mechanisms of coagulation factor Ⅶa-induced colon cancer 8W620 cell proliferation and migration

Objective To investigate the mechanisms that coagulation factor Ⅶa promotes proliferation and migration of a colon cancer cell line (SW620 cells) in vitro. Methods The expression of interleukin 8 (IL-8), tissue factor (TF), caspase-7 and p-p38 MAPK in SW620 cells treated with factor Ⅶa or protease activated receptor 2 aganist (PAR2-AP) was measured by ELISA, Western-blotting and QT-PCR. Results Factor Ⅶa and PAR2-AP induced IL-8 expression at both mRNA and protein levels, up-regulated TF mRNA expression and TF activity, but down-regulated caspese-7 mRNA and p-p38 MAPK levels in SW620 cells. The effects of factor Ⅶa were not only blocked by anti-TF but also by anti-PAR2 antibodies. Conclusion Factor Ⅶa binds to TF on cell surface, forming a complex which activates PAR2, then provoking IL-8 and TF expression, and suppresses caepase-7 expression, thus promotes the tumor cell proliferation and migration, p38 MAPK may negatively regulate this process.     

表没食子儿茶素没食子酸酯诱导人胃癌细胞裸鼠移植瘤凋亡的研究

背景与目的探讨绿茶提取物表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG)诱导人胃癌细胞裸鼠移植瘤凋亡及分子机制。方法建立人胃腺癌(SGC7901)细胞裸鼠异种移植瘤模型,用不同剂量的EGCG进行治疗,并设对照,用流式细胞分析术检测肿瘤组织中细胞凋亡情况,免疫组织化学方法检测肿瘤组织的凋亡相关基因Bcl-2、Bax、Caspase-3的表达情况。结果裸鼠异种移植瘤治疗实验结果显示,EGCG对移植瘤生长有明显抑制作用(其中20mg/kg、EGCG抑制率54.64%与对照组比较P<0.05);PI染色FCM分析发现,EGCG20mg/kg能诱导移植瘤细胞凋亡率17.2%与对照组比P<0.05;SP免疫组织化学结果表明EGCG可上调移植瘤细胞中Bax、Caspase-3蛋白的表达,下调Bcl-2蛋白的表达。结论表没食子儿茶素没食子酸酯(EGCG)具有体内诱导人胃腺癌细胞凋亡的作用,其机制可能与Bcl-2/Bax比值降低导致Caspase-3的活化从而启动细胞凋亡有关。     

玻连蛋白促进肝癌细胞株增殖及迁移的初步研究

目的： 了解玻连蛋白(VTN)对肝肿瘤细胞株SMMC7721增殖和迁移能力的影响。方法： 采用不同浓度VTN包被培养皿后接种、培养细胞,采用激光共聚焦显微镜观察VTN包被后细胞骨架成分微管蛋白形态的变化,用细胞增殖试剂分析VTN对细胞增殖率的影响及分析VTN对凋亡诱导剂抑制增殖作用的影响,采用Transwell侵袭实验检测VTN对细胞迁移能力的影响。结果：激光共聚焦观察结果显示VTN包被有助于促进细胞贴壁及细胞骨架蛋白形态结构的维持;VTN可促进肿瘤细胞的生长且呈剂量效应,并可减弱凋亡诱导剂对细胞增殖的抑制;Transwell侵袭实验结果显示VTN包被可促进细胞迁移。结论：体外实验条件下,VTN有助于肝癌细胞株SMMC7721的增殖及迁移,提示VTN在肝癌发生过程中可能具有促进肿瘤细胞增殖和迁移的生物学作用。     

瘦素对乳腺癌生长细胞动力学和形态学影响的研究

目的：研究瘦素对乳腺癌细胞株MCF-7细胞生长的作用。方法：分别使用0、25、50、100和200nmol／L瘦素作用于细胞株MCF-7细胞，同时在上述作用后24、48和72h采用光学显微镜观察细胞形态学改变，采用MTT比色方法，观察不同浓度瘦素对MCF-7细胞生长刺激的剂量、时间效应，比较在相对应剂量胰岛素情况下，二者对细胞增殖刺激作用的强弱。结果：加用瘦素各组细胞比未加瘦素细胞密度增加，形态变异。与正常对照组相比，各浓度瘦素（25、50、100和200nmol／L）均可明显促进MCF-7细胞增殖，其中50、100和200nmol／L瘦素作用24、48和72h后差异均有统计学意义，P〈0．05。瘦素对MCF-7细胞的增殖作用呈现一定的时间和剂量依赖效应。胰岛素各相对应组之间与瘦素比较，细胞增殖刺激作用差异无统计学意义，P〉0．05。结论：瘦素对乳腺癌细胞株MCF-7细胞增殖有刺激作用，且这种作用呈现一定的剂量、时间依赖效应，结果提示瘦素是促进乳腺癌细胞增殖的因素之一。     

瘦素对乳腺癌生长细胞动力学和形态学影响的研究

目的:研究瘦素对乳腺癌细胞株MCF-7细胞生长的作用。方法:分别使用0、25、50、100和200nmol/L瘦素作用于细胞株MCF-7细胞,同时在上述作用后24、48和72h采用光学显微镜观察细胞形态学改变,采用MTT比色方法,观察不同浓度瘦素对MCF-7细胞生长刺激的剂量、时间效应,比较在相对应剂量胰岛素情况下,二者对细胞增殖刺激作用的强弱。结果:加用瘦素各组细胞比未加瘦素细胞密度增加,形态变异。与正常对照组相比,各浓度瘦素(25、50、100和200nmol/L)均可明显促进MCF-7细胞增殖,其中50、100和200nmol/L瘦素作用24、48和72h后差异均有统计学意义,P<0.05。瘦素对MCF-7细胞的增殖作用呈现一定的时间和剂量依赖效应。胰岛素各相对应组之间与瘦素比较,细胞增殖刺激作用差异无统计学意义,P>0.05。结论:瘦素对乳腺癌细胞株MCF-7细胞增殖有刺激作用,且这种作用呈现一定的剂量、时间依赖效应,结果提示瘦素是促进乳腺癌细胞增殖的因素之一。     

蜂胶黄酮PB3A对大肠癌细胞SW480中RGS2和GEM基因表达水平的影响

目的 研究蜂胶黄酮(pinobanksin-3-acetate,PB3A)对体外培养的大肠癌SW480细胞中G蛋白信号转导调节因子2 (regulator of G-protein signaling 2,RGS2)和GTP结合丝裂原诱导蛋白(GTP binding protein overexpressed in skeletal muscle,GEM)基因转录和蛋白质表达水平的影响,探讨PB3A的抗肿瘤作用机制及其相关信号通路.方法 通过实时荧光定量RT-PCR和蛋白印迹法检测100 mg/L PB3A作用下人大肠癌细胞SW480中RGS2和GEM基因的转录和表达情况.结果 100 mg/L PB3A药物的干预诱导SW480细胞出现悬浮的细胞碎片、细胞体缩小、变圆、皱缩.药物干预之后,SW480细胞中RGS2和GEM基因在mRNA和蛋白水平上调表达.实验组与对照组比较,RGS2和GEM基因的mRNA转录水平差异有统计学意义(P＜0.01),蛋白质表达水平差异有统计学意义(P＜0.05).根据RT-PCR检测结果,利用Spearman相关性分析检测出RGS2和GEM高度正相关(r=0.929,P＜0.01).结论 PB3A干预大肠癌SW480细胞后使RGS2和GEM基因在转录和翻译水平上上调表达.PB3A的抗大肠癌作用机制可能与RGS2和GEM基因的上调表达有关.     

EGCG对人乳腺癌MDA-MB-435细胞株细胞增殖的抑制及机制

目的：研究绿茶提取物表没食子儿茶素—3—没食子酸酯(epigalloeateehin—3 gallste，EGCG)对人乳腺癌细胞株细胞周期的影响及机制。方法：利用细胞增殖测定试剂盒(Cell Proliferation Assay kit)来制作细胞生长曲线；流式细胞仪分析EGCG作用于人乳腺癌MDA—MB—435细胞株前后细胞周期的变化；RT—PCR及Western印迹法研究肿瘤细胞株加药干预前后细胞周期抑制因子p21^waf1/cip1的mRNA及蛋白表达的变化。结果：EGCG可明显抑制乳腺癌细胞MDA—MB—435细胞的增殖活性，40μg／ml EGCG干预后，MDA-M-B435细胞主要阻滞于G0／G1期，在24小时阻滞最为明显。在48及72小时也有阻滞。对照组G0／G1期的比例49．92％，40μg／ml EGCG干预24、48及72小时后G0／G1期的比例分别为71．56％、67．20％和61．59％。EGCG 40μg／ml处理肿瘤细胞株细胞后24小时可检测p21 mRNA和蛋白表达提高。结论：绿茶有效成分可抑制乳腺癌细胞的增殖，这可能与其诱导p21表达从而抑制细胞周期的时相转换有关，这表明绿茶可能在乳腺癌的治疗上具有一定的前景。     

TLR4基因多态性对肝癌细胞HepG2增殖、迁移及凋亡的影响

目的:研究TLR4基因多态性对人肝癌细胞HepG2细胞增殖、迁移及凋亡的影响,并探讨其相关分子机制.方法:构建TLR4野生型(WT)、1196(C/T)位点及896(A/G)位点突变型质粒并将其稳转HepG2细胞株中;CCK-8法检测、Annexin V-PE/7-AAD流式细胞仪及Transwell小室实验检测TLR4基因多态性对HepG2细胞增殖、迁移及凋亡的影响;Western blot检测NF-κB p65(nuclear factorκB p65)表达水平的差异.结果:Western blot结果表明三组TLR4过表达细胞株中TLR4的含量显著高于正常组.与正常HepG2细胞相比,三组TLR4过表达细胞株增殖能力、迁移能力及p65表达量均增加;与TLR4野生型组相比,两组突变型细胞株增殖能力、迁移能力及p65表达量均减弱,且凋亡率增加,差异具有统计学意义(P<0.05).结论:TLR4可能通过影响NF-κB p65相关蛋白的表达促进肝癌细胞HepG2的增殖及迁移,其基因1196(C/T)位点及896(A/G)位点的突变会减弱肝癌细胞的增殖及迁移能力.     

紫杉醇脂质体对卵巢癌细胞生长抑制作用的实验研究

目的分析紫杉醇脂质体对卵巢癌SKOV-3细胞的生长抑制作用,观察药物作用的时间-浓度关系,探究药物作用机制。方法应用不同剂量紫杉醇脂质体对SKOV-3细胞进行处理,通过MTT检测细胞存活率,倒置显微镜观察细胞形态,流式细胞术检测细胞凋亡作用及其对细胞周期的阻滞状态,Western blot分析Bcl-2基因蛋白表达情况。利用SPSS 13.0对数据进行统计学分析。结果紫杉醇脂质体对SKOV-3细胞有明显的剂量-时间依赖效应。紫杉醇与紫杉醇脂质体均可将SKOV-3细胞阻滞在G2/M期,且随药物浓度增加,凋亡细胞所占比例逐渐增加。通过Western blot方法发现,紫杉醇脂质体作用后SKOV-3细胞Bcl-2蛋白表达明显下降,Bax蛋白表达明显上调,Bax/Bal-2比例明显上调。结论紫杉醇以脂质体为转运载体后,并未改变其对卵巢癌SKOV-3细胞的抑制作用及作用周期,具有较好的临床应用前景。     

miR-200b对人结肠癌细胞增殖和迁移的影响

目的 研究miR-200b对人结肠癌sw620细胞E-cadherin表达的调节作用,探讨miR-200b对sw620细胞增殖及迁移的影响.方法 采用实时PCR (RT-PCR)技术分别检测转染pEGP-miR-200b后24h和72 h sw620细胞中miR-200b的表达变化;采用RT-PCR技术和Western blot方法检测转染miR-200b后对E-cadherin表达的影响;采用四甲基偶氮唑蓝法(MTT)和划痕擦伤实验检测转染miR-200b对sw620细胞增殖、迁移的影响.结果 miR-200b转染sw620细胞48 h和72 h后,miR-200b相对表达量较对照组显著上调(t=11.579,P＜0.01;t=11.579,P＜0.01);且抑制了细胞的增殖能力,第3天抑制作用最显著,抑制率为55.34％;细胞的迁移速度受到明显的抑制,两组在24、48、72 h的迁移距离有显著差异(t=11.579,P＜0.01;t=10.419,P＜0.01;t=6.955,P＜0.01).同时细胞中E-cadherin基因的mRNA和蛋白表达水平显著升高(t=10.432,P＜0.01;t=8.325,P＜0.01).结论 上调miR-200b表达可抑制结肠癌细胞的生物活性,该作用可能与E-cadherin的表达有关.     

β肽及其多聚物对人肝癌细胞株与基质黏附的抑制作用

目的研究β肽及其多聚物对肝癌细胞株与基质黏附的抑制作用.方法观察化学合成的β肽（β肽）、化学合成的二聚β肽（β2肽）、化学合成的三聚β肽（β3肽）、用大肠杆菌表达系统表达的表达β3肽及GRGDS对人肝癌细胞株SMMC-7721细胞及人肝癌高转移细胞株HCCLM6细胞与纤连蛋白（fibronectin,FN）的黏附作用的影响.结果各种多肽对细胞与FN的黏附均具有特异的抑制作用,呈现剂量效应相关关系和时间效应相关关系（P〈0.05）.且随着多肽重复次数的增多,对细胞与FN黏附的抑制作用越强,表达β3肽和β3肽的抑制肿瘤细胞黏附的作用最强,β2肽和GRGDS的作用次之,β肽的抑制作用最弱.各种多肽对HCCLM6细胞与FN黏附的抑制作用强于对SMMC-7721细胞的黏附抑制作用.结论表达β3肽对肝癌细胞株与基质的黏附具有强大的抑制作用,可能成为抗肿瘤转移的新的药物手段.