碱性成纤维细胞生长因子对庆大霉素致肾小管上皮损伤的拮抗效应

背景：体内实验证实成纤维细胞生长因子能有效保护庆大霉素致肾小管上皮细胞的损伤,但对体外培养细胞的作用如何不多见。目的：在建立庆大霉素肾毒性体外细胞模型的基础上,观察不同浓度碱性成纤维细胞生长因子对庆大霉素肾毒性的保护作用。方法：采用酶加网筛方法分离纯化昆明小鼠肾小管上皮细胞,调整细胞浓度为1×108 L-1,将细胞悬液移入96孔细胞培养板,分组培养：空白对照组：正常培养;庆大霉素组: 10,30,50 µL/孔 (即400,1 200,2 000 U/孔)记为G1、G2、G3;碱性成纤维细胞生长因子组：20,50,80 µL/孔(即90,225,360 ng/孔)记为B1、B2、B3;庆大霉素加碱性成纤维细胞生长因子干预组：先加碱性成纤维细胞生长因子12 h后,再加庆大霉素12 h培养,分9个剂量组,即G1B1、G1B2、G1B3、G2B1、G2B2、G2B3、G3B1、G3B2、G3B3,每组4复孔。观察细胞形态及数量变化。结果与结论：庆大霉素对肾小管上皮细胞的损伤呈剂量依赖性,中、高浓度组的上皮细胞皱缩,变圆,肿胀,贴壁差,内部胞质破坏严重,结构紊乱,低浓度组细胞数量改变不明显,并且开始有成纤维细胞出现;碱性成纤维细胞生长因子各组细胞饱满、折光性强,数量明显增多,50 µL/孔浓度以上效果显著,与80 µL/孔差异无显著性意义;庆大霉素加碱性成纤维细胞生长因子干预组中低浓度庆大霉素组细胞未见明显损害,细胞数量反而增多,中浓度庆大霉素组损害的细胞崩解减少、细胞皱缩和贴壁差的程度有所减轻,高浓度碱性成纤维细胞生长因子干预后细胞形态良好,但高浓度庆大霉素所致细胞肿胀、坏死损伤任何浓度的碱性成纤维细胞生长因子干预都无法改善。50 µL/孔碱性成纤维细胞生长因子对中、低浓度庆大霉素所致肾毒性有拮抗作用,对高浓度庆大霉素所致肾毒性无保护作用。     

转染碱性成纤维细胞生长因子基因在成肌细胞中表达及调控系统的建立

背景：作者前期研究已经成功将碱性成纤维细胞生长因子(basic fibroblast grouth factor, bFGF)基因转入鼠眼外肌的肌卫星细胞中,证明其能够在眼外肌的成肌细胞中表达并促进细胞增殖,促进其分化能力。目的：进一步探讨转染后碱性成纤维细胞生长因子基因在成肌细胞中表达的调控方法。方法：将目的基因碱性成纤维细胞生长因子与诱导表达载体pcDNA4/TO/myc-HisTMA连接,通过经菌落PCR和酶切鉴定的阳性克隆测序和EcoRⅠ and Hind Ⅲ双酶切处理和Xho Ⅰ单酶切处理验证。筛选并确定C2C12成肌细胞的抗生素的敏感性。通过脂质体转染技术,建立C2C12稳定表达pcDNA6/TR细胞系,经Western blot (蛋白印迹法)鉴定。将pcDNA4/TO/myc-孙HisTMA-bFGF转染到pcDNA6/TR-C2C12细胞系,免疫荧光法及Western blot法检测碱性成纤维细胞生长因子在四环素诱导的转染了pcDNA4/TO/myc-HisTMA-bFGF的C2C12细胞内的表达及其分泌情况,并设对照。结果与结论：①经测序对照,双酶及单酶切处理均证实碱性成纤维细胞生长因子与诱导表达载体pcDNA4/TO/myc-HisTMA成功连接。②blasticidin对C2C12细胞的最小致死浓度为10 mg/L,zeocin对C2C12细胞的最小致死质量浓度为750 mg/L。③建立的pcDNA6/TR- C2C12细胞系正确。④经四环素处理的转染了pcDNA4/TO/myc-HisTMA- bFGF的成肌细胞基因表达阳性,未经处理的则为阴性;经四环素处理的转染了pcDNA4/TO/myc- HisTMA-bFGF的成肌细胞产生碱性成纤维细胞生长因子蛋白,24 h达高峰,未处理的则不能产生碱性成纤维细胞生长因子蛋白。结果提示应用四环素抑制调节系统,可以调节碱性成纤维细胞生长因子基因在成肌细胞中的表达。     

碱性成纤维细胞生长因子和Noggin诱导人羊水来源干细胞向神经细胞分化的差异比较***☆

背景：相对于成体干细胞和胚胎干细胞自身存在的问题,羊水来源的干细胞系的建立,能为每一个个体建立一份自身的、具有高度增殖分化能力的干细胞储备,有望成为神经性退行性疾病细胞治疗的理想细胞来源。 目的：观察Noggin和碱性成纤维细胞生长因子对人羊水来源干细胞向神经细胞分化的影响。 方法：羊水标本来自怀孕16~22周行产前诊断的孕妇,在超声引导下行羊膜腔穿刺取得。利用CD117抗体,选用免疫磁珠法从人孕中期羊水标本中分离获得羊水干细胞,培养扩增后,通过流式细胞仪检测表面抗原表达进行鉴定。选取生长状态良好的第3代羊水干细胞,利用无血清的神经诱导培养液诱导其向神经细胞分化,分为空白对照组、基础诱导液组、Noggin诱导组和碱性成纤维细胞生长因子诱导组。倒置相差显微镜观察诱导后细胞形态的变化,细胞免疫荧光检测巢蛋白、β-Ⅲ tubulin和神经丝蛋白在诱导后细胞中的表达。 结果与结论：利用免疫磁珠方法分离出的羊水干细胞呈CD44和HLA-ABC表达阳性,CD45和HLA-DR表达阴性。诱导2周后,碱性成纤维细胞生长因子诱导组光镜下细胞形态发生显著变化,免疫荧光染色巢蛋白、β-Ⅲ tubulin和神经丝蛋白表达阳性率较高。Noggin诱导组细胞形态和染色结果与基础诱导液组无显著性差异。提示利用羊水来源的干细胞诱导分化为神经细胞的过程中碱性成纤维细胞生长因子的作用远优于Noggin。     

生骨注射液对骨折愈合过程中碱性成纤维细胞生长因子和血管内皮生长因子表达的影响

背景：在促进骨折修复研究中,应用外源性生长因子极不稳定,且造价高不适宜广泛推广,如何有效地促进内源性生长因子的表达将是值得深入研究的课题。 目的：观察生骨注射液对骨折愈合过程中内源性碱性成纤维细胞生长因子和血管内皮生长因子表达的影响。 设计、时间及地点：完全随机分组设计,对照实验,于2007-07/2008-08在华中科技大学同济医学院附属同济医院骨科实验室完成。 材料：清洁级3月龄雄性SD大鼠60只。生骨注射液由当归、土鳖虫、淫羊藿等药物组成,由湖北省中医院提供,质量浓度为1 g/L。 方法：建立SD大鼠胫骨干骨折愈合模型,分成实验组和对照组各30只,分别在骨折断端等量注射生骨注射液和生理盐水,每两日1次,0.2 mL/次。两组分别于术后第1,2,3,4,5,6周时各处死5只大鼠,取材。 主要观察指标：采用免疫组织化学方法,检测两组大鼠骨折愈合过程中不同阶段骨痂组织中碱性成纤维细胞生长因子、血管内皮生长因子蛋白的表达及差异情况。 结果：免疫组织化学染色显示,各时间点实验组骨痂组织中碱性成纤维细胞生长因子、血管内皮生长因子表达量及阳性定位均明显强于对照组。 结论：生骨注射液能够增加骨折愈合过程中碱性成纤维细胞生长因子、血管内皮生长因子在骨痂组织中的表达,这可能是生骨注射液促进骨折愈合的机制之一。     

重组人骨形态发生蛋白2与碱性成纤维细胞生长因子联合促进兔骨髓基质细胞血管内皮细胞生长因子表达及其成骨潜能

背景：了解在体外或体内环境下,应用重组人骨形态发生蛋白2和碱性成纤维细胞生长因子,能否达到既加强成骨又促进血管内皮细胞生长因子表达的双重作用。 目的：观察兔骨髓基质细胞经重组人骨形态发生蛋白2与碱性成纤维细胞生长因子刺激后,其血管内皮细胞生长因子表达及成骨潜能的变化。 方法：取兔双侧股骨骨髓基质细胞,采用骨髓基质细胞体外培养技术,单独或联合以重组人骨形态发生蛋白2、碱性成纤维细胞生长因子刺激细胞。细胞培养5 d后,进行细胞形态、增殖情况、碱性磷酸酶活性、成骨结节、血管内皮细胞生长因子阳性细胞率等项目的检测。 结果与结论：联合先后应用重组人骨形态发生蛋白2、碱性成纤维细胞生长因子在细胞计数、碱性磷酸酶活性、矿化面积百分率、血管内皮细胞生长因子阳性细胞率4个检测项目上优于同时应用重组人骨形态发生蛋白2或碱性成纤维细胞生长因子以及单独应用重组人骨形态发生蛋白2或碱性成纤维细胞生长因子。结果表明合理的联合使用重组人骨形态发生蛋白2和碱性成纤维细胞生长因子,不仅可促进骨髓基质细胞的快速增殖及向成骨细胞转化,还可促进促血管内皮细胞增生的重要介质血管内皮细胞生长因子的表达。     

RhoA/ROCK-I信号通路与增生性瘢痕成纤维细胞结缔组织生长因子的表达

背景：前期实验表明增生性瘢痕中RhoA和ROCK-I基因表达较正常皮肤高,提示RhoA/ROCK-I信号通路可能参与了增生性瘢痕的发生,但其在病理性瘢痕中的作用尚不清楚。 目的：研究RhoA/ROCK-I信号通路在增生性瘢痕成纤维细胞结缔组织生长因子(connective tissue growth factor,CTGF)表达调控中的作用。 方法：分离培养人增生性瘢痕组织来源的成纤维细胞,应用转化生长因子β1及Rho激酶的特异抑制剂Y-27632对细胞进行干预实验。采用实时荧光定量PCR及免疫荧光细胞化学方法检测瘢痕成纤维细胞中RhoA,ROCK-I及CTGF mRNA与蛋白的表达。 结果与结论：给予转化生长因子β1后,增生性瘢痕成纤维细胞中RhoA,ROCK-I及CTGF mRNA与蛋白表达明显增多(P < 0.01);而Y-27632能阻碍转化生长因子β1的作用;但单独给予Y-27632并不引起瘢痕成纤维细胞中RhoA,ROCK-I及CTGF的mRNA与蛋白表达改变。说明转化生长因子β1可通过RhoA/ROCK-I信号通路调控CTGF mRNA与蛋白的表达,即RhoA/ROCK-I信号通路参与了瘢痕成纤维细胞CTGF的表达调控,阻断RhoA下游通路是增生性瘢痕治疗靶点之一。     

中医外治法干预运动创伤性膝关节炎模型兔膝关节滑膜中碱性成纤维细胞生长因子的表达★

目的：实验拟验证外治法治疗运动创伤性关节炎模型兔膝关节滑膜中碱性成纤维细胞生长因子的表达，探讨中医外治法治疗运动创伤性关节炎的可能机制。 方法：实验于2006-01/06在广州体育学院和南方医科大学内分泌实验室完成。①实验分组：普通级健康雄性新西兰大耳白兔24只，体质量2.1~2.6 kg；随机数字表法分为正常组、模型组和外治法组，每组8只。②实验方法：以Hulth模型制备运动创伤性关节炎模型。造模8周后外治法组用中药熏蒸配合手法弹拨以及电刺激治疗4周。③实验评估：4周后每组取部分切片行常规苏木精-伊红染色；另每组取部分石蜡切片行免疫组织化学SABC法观察实验动物膝关节滑膜碱性成纤维细胞生长因子的表达。 结果：纳入健康雄性新西兰大耳白兔24只，均进入结果分析。①滑膜组织结构变化：正常组滑膜无增生肥厚，滑膜组织苏木精-伊红染色见纤维细胞排列均匀、规则；关节软骨外观呈蓝白色，无裂纹及溃疡。模型组滑膜充血、部分粘连，苏木精-伊红染色见滑膜绒毛肥厚与纤维样化；关节软骨失去原有的光泽。外治法组的关节软骨皆仍有光泽，略发黄，软骨表面无明显的裂纹、糜烂及溃疡形成。②滑膜组织中细胞因子变化：碱性成纤维细胞生长因子免疫组织化学检测平均吸光度正常组为0.126 1±0.029 0，模型组为0.238 6±0.040 0，与外治法组0.513 5±0.038 0相比较，差异显著（P < 0.01）。 结论：外治法能够促进实验动物膝关节滑膜细胞碱性成纤维细胞生长因子的表达，以及受损软骨的修复。     

丹参酮ⅡA对心脏成纤维细胞转化生长因子β1/Smads信号通路的作用

背景：转化生长因子β1通过Smads信号通路刺激心脏成纤维细胞增殖与分化是心肌纤维化最重要的发生机制之一。 前期研究证实丹参酮ⅡA能有效抑制心肌纤维化,但是否通过阻断转化生长因子β1/Smads信号通路起作用尚不清楚。目的：观察丹参酮ⅡA对大鼠心脏成纤维细胞内转化生长因子β1信号转导的影响。 方法：采用胰酶消化法和差速贴壁法获取新生SD大鼠心脏成纤维细胞,应用5 μg/L转化生长因子β1刺激及不同浓度丹参酮ⅡA (10-6,10-5和10-4 mol/L)。用反转录聚合酶链反应法和免疫蛋白印迹法分别检测转化生长因子β1刺激后6,12和24 h纤维连接蛋白的表达,免疫蛋白印迹法检测转化生长因子β1刺激后15,30,60和120 min的Smads蛋白表达。 结果与结论：纤维连接蛋白mRNA和蛋白表达量在转化生长因子β1刺激6 h后开始呈现上升趋势,至作用24 h时分别增加1.3倍和1.8倍(P < 0.01);磷酸化Smad2/3 蛋白表达量在转化生长因子β1刺激15 min后开始上升,1 h达到高峰,2 h后虽有所下降,但仍较刺激前增加3.9倍(P < 0.01)。丹参酮ⅡA(10-5和10-4 mol/L)预处理可下调纤维连接蛋白和磷酸化Smad2/3表达(P < 0.05或P < 0.01),而且效应呈剂量依赖性。由此可知,转化生长因子β1在一定范围内以时间依赖方式诱导纤维连接蛋白及其mRNA 和磷酸化Smad2/3表达。丹参酮ⅡA抗心肌纤维化作用可能与其抑制转化生长因子β1诱导的Smad2/3磷酸化,阻断心脏成纤维细胞内转化生长因子β1/Smads信号通路有关。     

碱性成纤维细胞生长因子荧光真核表达载体构建及对过氧化氢诱导血管内皮细胞凋亡的影响

背景：已证实外源性碱性成纤维细胞生长因子(basic fibroblast growth factor , bFGF)可抑制血管内皮细胞凋亡。 目的：构建表达bFGF的荧光真核表达载体,探讨其对过氧化氢(H2O2)诱导的血管内皮细胞凋亡和凋亡相关蛋白的影响。 方法：通过基因亚克隆构建荧光真核表达载体pcDNA3.1-bFGF-GFP,利用脂质体介导将bFGF 基因导入人脐静脉内皮细胞内,通过荧光观察和RT-PCR检测基因的表达。实验分为3组,对照组(转染pcDNA3.1)、过氧化氢组(转染pcDNA3.1+ H2O2)和bFGF转染+过氧化氢组(转染pcDNA3.1-bFGF-GFP+H2O2),流式细胞术测定细胞凋亡率,Western blot检测caspase-3 P17活性亚单位和Bax蛋白表达。 结果与结论：成功构建荧光真核表达载体pcDNA3.1-bFGF-GFP,该载体转染人脐静脉内皮细胞后,bFGF mRNA显著增加,并可观察到绿色荧光。与对照组相比,过氧化氢组细胞凋亡率和caspase-3 P17活性亚单位、Bax蛋白的表达量都明显增加(P < 0.01),而bFGF 转染+过氧化氢组的细胞凋亡率和caspase-3 P17活性亚单位、Bax蛋白的表达量则比过氧化氢组显著降低(P < 0.01)。证实bFGF基因转染能抑制过氧化氢诱导的血管内皮细胞凋亡,其作用机制可能与调控Bax蛋白表达和caspase-3活性有关。     

碱性成纤维细胞生长因子对大鼠脑出血血肿周围脑组织和血浆中血栓素B2和6-酮-前列腺素F1a含量的影响

目的观察大鼠脑出血后应用碱性纤维细胞因子(bFGF)对血肿周围脑组织及血浆中TXB2和6-K-PGF1a含量及比值的影响。方法采用脑内注射胶原酶建立大鼠脑出血模型,分为脑出血模型对照组和bFGF治疗组,并于脑内注射生理盐水建立假手术组对照组。每组又分为1d和3d两个时间点,bFGF治疗组应用bFGF进行治疗,检测1d和3d后血肿周围脑组织及血浆中TXB2和6-K-PGF1a含量。结果bFGF治疗组与模型对照组比较,1d时血浆中TXB2含量降低(P<0.05),1d和3d时6-K-PGF1a含量则均升高(P<0.05),TXB2/6-K-PGF1a比值1d(P<0.05)和3d(P<0.01)时均降低;bFGF治疗组与模型对照组比较,血肿周围脑组织中TXB2含量3d时较脑出血模型组升高(P<0.01),6-K-PGF1a含量无显著差异(P>0.05),TXB2/6-K-PGF1a比值1d时降低(P<0.05),3d时升高(P<0.05)。结论脑出血后血浆中TXB2和6-K-PGF1a含量的变化比血肿周围脑组织中明显;bFGF可使大鼠脑出血后血浆中TXB2含量减少,6-K-PGF1a含量增多,TXB2/6-K-PGF1a比值降低。     

Synergistic induction of t-PA by vascular endothelial growth factor and basic fibroblast growth factor and localization of t-PA to Weibel-Palade bodies in bovine microvascular endothelial cells

Endothelial cell migration is stimulated by members of the vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) families, and is dependent on extracellular proteolytic activity provided by enzymes of the plasminogen activator (PA) system. Here we report that in bovine microvascular endothelial cells (BME cells), bFGF principally increased urokinase-type PA (u-PA) while tissue-type PA (t-PA) was increased mainly by VEGF. In bovine aortic endothelial cells (BAE cells), bFGF increased u-PA, whereas VEGF had no effect. Co-added bFGF and VEGF increased t-PA mRNA levels and enzyme activity in both cell types in a synergistic manner. Tissue-type plasminogen activator (t-PA) immunoreactivity colocalized with von Willebrand factor, a marker for Weibel-Palade bodies. Co-added bFGF and VEGF increased the number of t-PA-positive cells as well as the number of t-PA-positive granules per cell. Localization of t-PA in regulated storage granules endows endothelial cells with the potential to rapidly increase proteolytic activity in the pericellular environment.     

Differential induction of nerve growth factor and basic fibroblast growth factor mRNA in neonatal and aged rat brain

Stimulation of glucocorticoid or β-adrenergic receptors (BAR) has been shown to increase nerve growth factor (NGF) biosynthesis in adult rat brain. Little is known about the role of these receptors in the regulation of NGF expression in neonatal and aged brain. We have examined the effect of the synthetic glucocorticoid dexamethasone (DEX) and the BAR agonist clenbuterol (CLE) on the levels of NGF mRNA in neonatal (8 day old), adult (3 month old) and aged (24 month old) rats. By 3 h, DEX (0.5 mg/kg, s.c.) evoked a comparable increase in NGF mRNA in the cerebral cortex and hippocampus in both 8-day and 3-month-old rats. In contrast, CLE (10 mg/kg, i.p.) failed to change NGF mRNA levels in neonatal rats, while increasing (2–3-fold) NGF mRNA levels in the cerebral cortex of adult rats. In 24-month-old rats, both DEX and CLE elicited only a modest increase in NGF mRNA. This increase was, however, anatomically and temporally similar to that observed in adult animals. The weak effect of DEX or CLE was not related to a down-regulation of receptor function because both DEX and CLE were able to elicit a comparable increase in the mRNA levels for basic fibroblast growth factor (FGF2) in neonatal, adult and aged rat brain. Our data demonstrate that induction of NGF expression by neurotransmitter/hormone receptor activation varies throughout life and suggest that pharmacological agents might be useful tools to enhance trophic support in aging.     

Directional induction of dopaminergic neurons from neural stem cells using substantia nigra homogenates and basic fibroblast growth factor

To date, complex components of available reagents have been used for directional induction of neural stem cells into dopaminergic neurons, resulting in a poor ability to repeat experiments. This study sought to investigate whether a homogenate of the substantia nigra of adult rats and/or basic fibroblast growth factor could directionally induce neural stem cells derived from the subventricular zone of embryonic rats to differentiate into dopaminergic neurons. Tyrosine hydroxylase-positive cells were observed exclusively after induction with the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor for 48 hours in vitro. However, in the groups treated with homogenate supernatant or basic fibroblast growth factor alone, tyrosine hydroxylase expression was not observed. Moreover, the content of dopamine in the culture medium of subventricular zone neurons was significantly increased at 48 hours after induction with the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor. Experimental findings indicate that the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor could directionally induce neural stem cells derived from the subventricular zone of embryonic rats to differentiate into dopaminergic neurons in the substantia nigra with the ability to secrete dopamine.     

Neurogenesis in neonatal rat brain is regulated by peripheral injection of basic fibroblast growth factor (bFGF)

Many major diseases of human brain involve deficiencies of select neuronal populations. As one approach to repair, we examined regulation of neurogenesis directly in vivo, employing postnatal day 1 (P1) cerebellar cortex, which is composed primarily of granule neurons and dividing precursors. We focused on basic fibroblast growth factor (bFGF), which stimulates precursor mitosis in culture and which is highly expressed in cerebellum during neurogenesis. Subcutaneous injection of bFGF increased [³H]thymidine ([³H]dT) incorporation, a marker for DNA synthesis, by 50% in whole cerebellar homogenates, suggesting that peripherally administered factor altered ongoing neural proliferation. Further, assay of isolated granule precursors revealed a 4-fold increase in [³H]dT incorporation following in vivo bFGF treatment, indicating that granule neuroblasts were the major bFGF-responsive population. Morphologic analysis indicated that twice as many granule precursors were in S-phase of the mitotic cycle after peripheral bFGF. To determine whether other neurogenetic populations respond to peripheral bFGF, we examined additional brain regions in vivo. bFGF stimulated DNA synthesis by 68% in hippocampus, and by > 250% in pontine subventricular zone (SVZ). In contrast, incorporation was not altered in basal pons or cerebral cortex, regions in which neurogensis has already ceased. To define potential direct actions of peripherally administered factor, ¹²⁵I-bFGF was used to study distribution. Intact 18 kDa ¹²⁵I-bFGF was recovered from brain following peripheral injection, suggesting that the factor acted directly to stimulate mitosis in dividing neuroblasts. The stimulation of neuronal proliferation by exogenous bFGF suggests that the factor normally regulates neurogenesis, and provides new therapeutic approaches to promote functional recovery from nervous system diseases. © 1996 Wiley-Liss, Inc.     

外源性碱性成纤维细胞生长因子对缺血大鼠脑内源性碱性成纤维细胞生长因子表达的影响

目的 研究外源性碱性成纤维细胞生长因子(bFGF)缩小局灶性脑缺血梗死灶的机制。方法 用免疫组化ABC法检测在局灶性脑缺血模型上给予生理盐水或bFGF后早期生长反应蛋白-1(Egr-1)，bFGF，碱性成纤维细胞生长因子受体(bFGFR)的动态表达。结果 给药组在3h～3d各时间段梗死灶均有不同程度的缩小。对照组和给药组Egr-1表达均表现为3～6h的增强过程，但给药组更强于对照组。对照组12h见有bFGF表达增强，而bFGFR表达3h到达高峰，6h起下降，12h时bFGFR的表达已恢复至正常水平(出现了配体和受体表达时相上不匹配)。给药组bFGF表达提前且增强，3h即见有bFGF表达增强，6h时出现第一峰，从而与bFGFR 3～6h的表达增强过程相吻合。结论 外源性bFGF能缩小梗死灶，该神经保护作用是通过Egr-1蛋白高表达使内源性bFGF的表达增高且提前，从而与bFGFR的表达增强过程重叠而实现的。     

Tissue factor is regulated by epidermal growth factor in normal and malignant human endometrial epithelial cells

Tissue Factor (TF), the initiator of the extrinsic coagulation cascade, is overexpressed in a variety of cancers. TF is also expressed in normal human endometrium but little is known about its expression or regulation in endometrial cancer. We demonstrate herein that TF is expressed in the endometrial adenocarcinoma cell line Ishikawa. Furthermore, epidermal growth factor (EGF) induces a rapid and sustained increase in TF expression. Estradiol and progesterone had no effect on basal or EGF-induced TF expression in Ishikawa cells. In contrast to the pronounced and sustained upregulation at the protein level, EGF treatment elicited only a modest and transient increase in TF mRNA levels. This activity corresponded to the response observed from an exogenous TF promoter construct. However, the induction of TF was abrogated by cycloheximide as well as actinomycin-D, inhibitors or protein- and mRNA-synthesis, respectively, demonstrating that EGF mediates its effect through activation of the TF gene. Fractionation experiments showed that EGF increases TF presence in caveolin-I containing membrane fractions. Coagulation and invasion assays were used to explore the physiological implications of TF regulation. The results demonstrate that EGF-mediated induction of TF increases the procoagulant activity and invasive potential of Ishikawa cells. Furthermore, immunocytochemistry confirmed that TF is regulated by EGF in primary cultures of normal endometrial epithelial cells and malignant tumor cells. In conclusion, EGF-mediated upregulation of TF results in accumulation of this glycoprotein in caveolae-like membrane fractions and increased coagulative and invasive potential. Our results suggest that TF may play an integral role in endometrial carcinogenesis.