PPARγ激动剂对成人正常皮肤成纤维细胞转分化的作用

目的：观察过氧化物酶体增殖物激活受体γ（PPARγ）激动剂对转化生长因子β1（TGF-β1）诱导成纤维细胞转分化的影响,探讨其抗瘢痕纤维化的潜在作用。方法：体外培养成人正常皮肤成纤维细胞,利用免疫荧光细胞化学法观察、分析PPARγ配体15-脱氧前列腺素J2（15d-PGJ2）及其激动剂曲格列酮对TGF-β1诱导的α平滑肌动蛋白（α-SMA）表达的影响,利用Western blot、实时荧光RT-PCR技术检测PPARγ激动剂对TGF-β1诱导的α-SMA蛋白及mRNA水平的影响。结果：TGF-β1能显著增加成纤维细胞转分化为肌成纤维细胞;与TGF-β1诱导组相比,10μM曲格列酮、15d-PGJ2预处理组的α-SMA表达量显著减少（P〈0.01）,抑制效应分别为31%、57%;预处理组的α-SMA mRNA水平显著下降（P〈0.01）。结论：PPARγ激动剂能抑制TGF-β1诱导的成人正常皮肤成纤维细胞的转分化的效应,具有抗皮肤瘢痕化、挛缩的潜在作用。     

PPAR γ激动剂对TGF-β1诱导皮肤成纤维细胞细胞外基质表达的影响

目的:研究过氧化物酶体增殖物激活受体γ(PPARγ)激动剂对转化生长因子β1(TGF-β1)诱导人正常皮肤成纤维细胞细胞外基质(extracellular matrix,ECM)表达效应作用的影响,探讨其抗瘢痕的潜在作用。方法:体外培养人正常皮肤成纤维细胞,羟脯氨酸比色法观察PPARγ配体15-脱氧前列腺素J2(15-deoxy-Δ12,14-prostaglandin J2,15d-PGJ2)及其激动剂曲格列酮对TGF-β1诱导的胶原表达的影响,免疫细胞化学法及图像分析技术观察、分析PPARγ激动剂对TGF-β1诱导的纤维连接蛋白(fibronectin,FN)表达的影响,MTT法观察不同浓度的PPARγ激动剂对成纤维细胞增殖活性的影响。结果:TGF-β1能显著增加人正常皮肤成纤维细胞胶原和FN表达,并呈剂量依赖效应;与TGF-β1刺激组相比,10μM15d-PGJ2、曲格列酮预处理组胶原和FN表达减少,有显著性差异(P<0.01);PPARγ激动剂对成纤维细胞的增殖活性影响分析,各实验组与对照组相比无显著性差异(P>0.05)。结论:PPARγ激动剂可抑制TGF-β1诱导的人正常皮肤成纤维细胞ECM合成增多的效应,具有体外抗瘢痕纤维化的作用。     

过氧化物酶体增殖物激活受体γ激动剂对转化生长因子β1诱导皮肤成纤维细胞作用的影响

目的 观察过氧化物酶体增殖物激活受体γ(PPARγ)激动剂对转化生长因子(TGF)-β1诱导成纤维细胞(Fb)转分化及对胶原牛成作用的影响.方法 体外培养成人正常皮肤Fb,免疫荧光细胞化学法观察PPARγ配体15-脱氧前列腺素J2(15d-PGJ2)、曲格列酮对TGF-β1诱导的α平滑肌动蛋白(α-SMA)表达的影响,Western blot检测15d-PGJ2、曲格列酮对TGF-β1诱导的α-SMA及Ⅰ型胶原蛋白表达的影响,噻唑蓝(MTT)比色法观察对Fb增殖活性的影响.结果 与TGF-β1诱导组比较,10μmoL/L曲格列酮、10 μmol/L 15d-PGJ2预处理组的α-SMA表达量显著减少(P<0.01),抑制效应分别为31%、57%;预处理组的Ⅰ型胶原表达量也屁著减少(P<0.01),抑制效应分别为57%、38%.曲格列酮、15d-PGJ2对Fb的增殖活性影响分析,各实验组与对照组比较差异无统计学意义(P>0.05).结论 PPARγ激动剂能抑制TGF-β1诱导的人正常皮肤Fb的转分化和Ⅰ型胶原合成增多的效应,具有抗瘢痕的潜在作用.     

温阳消癥方调控LncRNA MEG3对TGF-β1诱导的肾小管上皮细胞纤维化因子的影响

目的:探讨温阳消癥方对TGF-β₁诱导的人近端肾小管上皮细胞LncRNA MEG3的调控作用及对纤维化因子的影响。方法:体外培养人近端肾小管上皮细胞(HK-2),分为对照组、TGF-β₁组、TGF-β₁+3.5 mg/ml温阳消癥组、TGF-β₁+14 mg/ml温阳消癥组，MTT法检测细胞增殖，实时荧光定量聚合酶链式反应(RT-qPCR)检测α-平滑肌肌动蛋白(α-SMA)、纤维连接蛋白(FN)及结缔组织生长因子(CTGF)mRNA及长链非编码RNA母系表达基因3(LncRNA MEG3)表达，蛋白质印迹法检测α-SMA、FN、CTGF蛋白表达。结果:3.5、7、14 mg/ml温阳消癥不影响细胞增殖，35 mg/ml组细胞增殖明显降低(P<0.01);与对照组比较，TGF-β₁组中α-SMA、FN、CTGF mRNA、LncRNA MEG3及FN、CTGF蛋白表达显著升高(P<0.01),α-SMA蛋白表达升高(P<0.05);与TGF-β₁     

血管内皮生长因子抑制肾小管上皮间充质转化及其与结缔组织生长因子和PI3K-Akt信号通路的关系

目的　探讨血管内皮生长因子（VEGF）对转化生长因子β1（TGF-β1）诱导的肾小管上皮间充质转化（EMT）的作用,及其与结缔组织生长因子（CTGF）、PI3K-Akt信号通路的关系。 方法 （1）将体外培养的HK2细胞分为正常对照组、TGF-β1（5 μg/L,下同）组、VEGF组（100 μg/L,下同）、TGF-β1＋VEGF组。HK2细胞体外培养48 h,用免疫组化双染方法检测肾小管上皮细胞α平滑肌肌动蛋白（α-SMA）和E钙黏蛋白的表达。（2）将体外培养的HK2细胞分为正常对照组、TGF-β1组、VEGF组、TGF-β1＋VEGF组、PI3K-Akt信号通路阻断剂LY294002组（25 μmol/L,下同）、TGF-β1＋LY294002组、VEGF＋LY294002组、TGF-β1＋VEGF＋LY294002组。HK2细胞体外培养48 h,用Western印迹和RT-PCR方法检测α-SMA和CTGF的表达;用ELISA方法检测培养上清中纤连蛋白（FN）和I型胶原（ColⅠ）的表达。 结果 免疫组化结果显示,TGF-β1组α-SMA表达比正常对照组增强,而E钙黏蛋白表达减弱; TGF-β1＋VEGF组α-SMA表达比TGF-β1组显著减弱,而E钙黏蛋白表达增强。 TGF-β1组α-SMA、CTGF蛋白和 mRNA及FN、ColⅠ表达比正常对照组显著增强（均P < 0.05）;TGF-β1＋VEGF组α-SMA、CTGF蛋白和mRNA及FN、ColⅠ表达比TGF-β1组显著减弱（均P < 0.05）;TGF-β1＋VEGF＋LY294002组α-SMA、CTGF蛋白和 mRNA及FN、ColⅠ表达比TGF-β1＋VEGF组显著增强（均P < 0.05）。 结论　VEGF能抑制TGF-β1诱导的体外培养的HK2细胞发生EMT,其机制可能与VEGF下调HK2细胞CTGF表达及减少细胞外FN、ColⅠ合成有关。VEGF的这种作用可能部分通过PI3K-Akt信号转导通路实现,其确切机制有待进一步研究。     

转化生长因子β1诱导肾间质成纤维细胞表型转化及其对结缔组织生长因子基因表达的影响

目的 研究转化生长因子β1（transforming growth factor beta-1,TGF-β1）诱导大鼠正常肾间质成纤维细胞（NRK-49F）表型转化过程中结缔组织生长因子（connective tissue growth factor,CTGF）基因表达的变化.方法 以不同浓度的TGF-β1（0、0.5、1、2、5、10 ng/ml）刺激NRK-49F细胞,分别应用MTT比色法、Western Blot、Northern Blot方法,检测TGF-β1刺激后肾间质成纤维细胞的增殖、Ⅰ型、Ⅲ型前胶原mRAN的表达、细胞表型标志物α-SMA mRNA及蛋白质表达、CTGFmRNA的表达变化.结果 1 ng/ml浓度以上TGF-β1能显著促进NRK-49F细胞增殖,上调Ⅰ、Ⅲ型前胶原mRNA的表达水平,诱导肌成纤维细胞表型标志α-SMA mRNA及蛋白质的表达,显著上调CTGF mRNA的表达水平（P＜0.01或P＜0.05）;以上效应均呈浓度依赖性.同时CTGF mRNA表达水平的增高与细胞表型转化的程度相一致.结论 TGF β1能诱导肾间质成纤维细胞发生表型转化,并促进了细胞增殖及细胞外基质的合成;该效应与TGF β1显著上调CTGF的基因表达一致.     

过氧化物酶体增殖物激活受体γ配体抑制大鼠肝纤维化的实验研究

目的 研究过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptorgamma,PPARγ)配体对大鼠肝纤维化的作用.方法 将Wistar大鼠40只随机分为两组,对照组(20只)和罗格列酮组(20只).所有动物使用饮水中加人质量比0.3‰硫代乙酰胺的方法 制作肝纤维化模型.对照组喂饲普通颗粒饲料.罗格列酮组喂饲含200 ppm罗格列酮的颗粒饲料.喂饲6个月后,用RT-PCR方法 检测肝纤维化大鼠肝脏PPARγ、TGF-β 1 及Ⅰ型前胶原mRNA表达,用Westernblot法检测PPARγ、TGF-β 1 、Ⅰ型胶原及α平滑肌肌动蛋白(α-SMA)表达,用Van Gieson(VG)染色的方法 检测肝组织切片的胶原表达情况.结果 罗格列酮组与对照组相比,PPARγmRNA表达显著增强(t=6.93,P<0.01),TGF-β 1 mRNA(t=3.89,P<0.01)和Ⅰ型前胶原mRNA表达显著降低(t=5.67,P<0.01).PPARγ、TGF-β 1 及Ⅰ型胶原蛋白表达所得结果 与RT-PCR结果 相一致.罗格列酮组与对照组相比,α-SMA表达显著降低(t=3.12,P<0.01).罗格列酮组肝组织切片的胶原染色低于对照组(t=3.47,P<0.01).结论 PPARγ配体能够抑制大鼠纤维化肝脏的胶原产生,在体内具有一定的抗肝纤维化作用.     

CKIP-1表达在前列腺部尿道少瘢痕愈合中的作用机制

目的探究酪蛋白激酶2相互作用蛋白1(CKIP-1)表达在前列腺部尿道少瘢痕愈合中的作用机制。方法收集小儿尿道瘢痕组织和正常尿道组织。通过酶消化法联合组织块法建立原代的人尿道瘢痕成纤维细胞。通过质粒转染沉默或过表达CKIP-1。通过qPCR和Western blot检测mRNA和蛋白的表达水平。CCK-8法检测细胞活力。免疫荧光染色检测α-SMA的表达。结果与正常尿道组织相比,瘢痕组织中CKIP-1水平降低而ROCK升高(P<0.05)。OE-CKIP-1组的CKIP-1mRNA和蛋白水平升高,细胞活力、α-SMA、COLⅠ、COLⅢ、TGF-β1和ROCK蛋白水平均显著低于对照组(P<0.05)。siCKIP-1组的CKIP-1mRNA和蛋白水平降低,其他上述指标均显著高于对照组(P<0.05)。结论 CKIP-1蛋白可能通过下调TGF-β1抑制ROCK2相关通路,并抑制COLⅠ、COLⅢ和α-SMA的表达,从而抑制人尿道瘢痕成纤维细胞纤维化。     

罗格列酮对转化生长因子β1诱导的人肾小管上皮细胞结缔组织生长因子表达的影响

目的探讨体外条件下转化生长因子(TGF)β1对人近端肾小管上皮细胞系(HK-2)表达结缔组织生长因子(CTGF)的诱导情况,以及罗格列酮对其影响和作用途径。方法应用RT-PCR方法检测CTGF和α-平滑肌肌动蛋白(SMA)mRNA表达。应用Western印迹方法检测CTGF和纤连蛋白(FN)的蛋白表达。结果(1)HK-2细胞低水平表达CTGFmRNA和蛋白,TGF-β1呈剂量和时间依赖性增加HK-2细胞CTGFmRNA和蛋白表达(P<0.01)。(2)罗格列酮和15脱氧前列腺素J2(15d-PGJ2)呈剂量依赖性抑制TGF—β1(4ng/ml)诱导的HK-2细胞CTGFmRNA和蛋白的表达。(3)过氧化物酶体增殖体激活受体(PPAR)γ特异性抑制剂GW9662(1μmol/L)完全阻断罗格列酮和15d—PGJ2对CTGFmRNA和蛋白表达的抑制作用。(4)与单纯TGF-β14ng/ml刺激组相比,罗格列酮5、10μmol/L处理组HK-2细胞FN蛋白分别下调39.9%和53.4%(P<0.01),并呈剂量依赖性(P<0.01),而GW9662完全阻断该作用。结论在体外,罗格列酮可通过激活肾小管上皮细胞PPARγ抑制TGF-β1诱导的CTGF转录和表达,同时也可抑制FN的表达。     

γ-干扰素对梗阻性积水肾间质纤维化的治疗作用

目的 探讨γ干扰素(IFN-γ)对梗阻性肾积水肾间质纤维化的抑制作用及其可能的机制.方法 将65只雄性SD大鼠随机分成4组:治疗组(20只)、模型组(20只)、药物对照组(20只)、假手术组(5只).在建模和给药后的第3、7、14、21、28天,每组各随机处死动物4只(假手术组1只).采用苏木素-伊红(HE)和Masson染色观察病变肾脏间质纤维化的情况;采用聚合酶链反应(RT-PCR)技术检测肾组织中转化生长因子-β1(TGF-β1)、α-平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原(Col-Ⅰ)的mRNA表达情况;免疫组织化学法观察以上三种蛋白的变化.结果 模型组大鼠术后肾间质逐渐出现纤维化改变,并随梗阻时间的延长逐渐加重,TGF-β1、α-SMA和Col-Ⅰ的mRNA和蛋白表达亦逐渐升高.第14天,模型组TGF-β1和α-SMA的表达量达到高峰,28 d时各指标的表达均达到最高,TGF-β1、α-SMA和Col-Ⅰ分别为51.84%、72.59%和68.73%.治疗组各指标在不同时间点均低于模型组,第28天时,三项指标依次为33.84%、32.59%和48.73%,与模型组比较P＜0.05.Banff评分显示治疗组间质纤维化程度明显减轻(P＜0.01).其余两组未见肾间质纤维化改变.结论 IFN-γ具有减轻积水后肾间质纤维化程度的作用,该作用与其下调TGF-β1的表达、抑制肌成纤维细胞(MyoF)激活和减少Col-Ⅰ生成有关.     

血管紧张素1-7对糖尿病大鼠肾小管间质纤维化的影响

目的 研究血管紧张素1-7(Ang1-7)对糖尿病大鼠肾小管间质纤维化的影响及其可能机制.方法 32只雄性Wistar大鼠被随机分为4组:健康对照组(NC组)、模型组(DM组)、替米沙坦组(TM组)、治疗组(T组).建模成功后第9周末检测各组大鼠24 h尿蛋白量、尿NAG/Cr、血糖、血胰岛素、三酰甘油(TG)、总胆固醇(TC)、BUN、Scr、血K+及血Na+ ;PAS染色观察肾脏病理改变 ;实时定量PCR法检测各组大鼠肾脏组织中转化生长因子β1(TGF-β1)、过氧化物酶体增殖物激活受体(PPAR)γ、α平滑肌肌动蛋白(α-SMA)mRNA水平 ;Western印迹法检测PPARγ、α-SMA、TGF-β1蛋白表达.结果 (1)第9周末,DM组大鼠血压、尿蛋白量、肾质量/体质量较NC组显著升高(P＜0.05),TM组及T组较DM组显著降低(P＜0.05),且T组变化更明显.(2)DM组第9周末肾间质损伤指数显著高于NC组(P＜0.05),TM组及T组则低于DM组(P＜0.05).(3)实时定量PCR结果显示,DM组TGF-β1、α-SMAmRNA水平显著升高(P＜0.05),PPARγ mRNA水平显著下降(P＜0.05),TM组及T组较DM组TGF-β1、α-SMA mRNA水平均显著下降(P＜0.05),PPARγ mRNA水平显著上升(P＜0.05),且T组变化更明显.(4)Western印迹结果显示,DM组TGF-β1、α-SMA蛋白水平显著升高(P＜0.05),PPARγ蛋白水平显著下降(P＜0.05),TM组及T组较DM组TGF-β1、α-SMA蛋白水平均显著下降(P＜0.05),PPARγ蛋白水平显著上升(P＜0.05),且T组变化更明显.结论 Ang1-7在体内可通过上调PPARγ表达,抑制α-SMA表达,对糖尿病大鼠肾小管间质纤维化可能具有抑制作用.     

TNF-α对兔胆管成纤维细胞P311/TGF-β1/α-SMA通路的影响及川芎嗪的干预作用

目的:探讨TNF-α对兔胆管成纤维细胞P311/TGF-β1/α-SMA通路的影响及川芎嗪的干预作用。方法:分离、培养正常家兔胆管成纤维细胞并鉴定,将胆管成纤维细胞分别给予TNF-α、TNF-α联合不同浓度的TMP(0.08、0.4、2.0 mg/m L)干预48 h,以无处理的胆管成纤维细胞为空白对照,用CCK-8法检测细胞增殖水平;real-time PCR检测细胞P311、TGF-β1、α-SMA m RNA表达;Western blot检测细胞TGF-β1、α-SMA蛋白表达。结果:与空白对照比较,胆管成纤维细胞经TNF-α处理后,增殖明显增强,P311、TGF-β1、α-SMA m RNA以及TGF-β1、α-SMA蛋白表达明显上调(均P0.05);TMP对TNF-α的上述效应有抑制作用,并且呈现浓度依耐趋势,其中0.4、2.0 mg/m L的TMP有明显作用(均P0.05)。结论:TNF-α可能通过调控P311/TGF-β1/α-SMA信号通路促进胆管成纤维细胞增殖,TMP能抑TNF-α对该通路的活化,故可能对良性胆道狭窄有防治作用。     

PPARγ特异配体抑制肝星状细胞的活化

目的 研究不同浓度的过氧化物酶体增殖物活化的受体γ(peroxisome proliferator-activated receptor gamma,PPARγ)特异配体罗格列酮对肝星状细胞(hepatic stellate cell,HSC)生物学特性的影响,以探究其在肝旱状细胞活化中的作用.方法 设立对照组,3μM罗格列酮组,10μM罗格列酮组,20μM岁格列酮组.用MTT法检测细胞的增殖情况;采用RT-PCR方法检测其中PPARγ、TGF-β1及Ⅰ型前胶原mRNA表达;用Western blot法检测PPARy、Ⅰ、Ⅲ型胶原及TGF-β1蛋白表达;用免疫细胞化学方法测定α-SMA表达的变化;ELISA法检测细胞培养上清中的Ⅰ型胶原表达的变化.结果 (1)RT-PCR:20μM罗格列嗣组或10μM罗格列酮组与3 μM罗格列酮组或对照组相比,PPARY mRNA表达显著增高(P＜0.01),Ⅰ型前胶原mRNA表达显著降低(P＜0.01);20 α-SMA罗格列酮组与10 α-SMA罗格列酮组之间,3 α-SMA罗格列酮组与对照组之间,PPARγ和Ⅰ型前胶原mRNA的表达差异无显著件(P＞0.05).而各组之间的TGF-β1 mRNA的差异无显著性意义(P＞0.05).(2)Western blot:PPARγ及TGF-β1蛋白表达所得结果与RT-PCR结果相一致.Ⅰ型胶原表达与RT-PCR Ⅰ型前胶原mRNA表达结果相一致.各组之间的Ⅲ型胶原表达差异无显著性意义(P＞0.05).(3)免疫细胞化学:20α-SMA罗格列酮组或10 α-SMA罗格列酮组与3 α-SMA罗格列酮组或对照组相比,α-SMA表达明显降低(P＜0.05).20 α-SMA岁格列酮组与10 α-SMA罗格列酮组之问,3 α-SMA罗格列酮组与对照组之间,差异无显著性(P＞0.05).(4)ELISA:20 α-SMA罗格列酮组或10 α-SMA罗格列酮组与3α-SMA罗格列倒组或对照组相比,细胞的培养上清中Ⅰ型胶原表达明显降低(P＜0.01).20 α-SMA罗格列酮组与10 α-SMA罗格列酮组之问,3 α-SMA罗格列酬组与对照组之间,差异无显著性(P＞0.05).结论 PPAR?配体罗格列酬能够在促进PPAR?的合成表达的同时,抑制细胞的增殖及胶原合成,抑制α-SMA的表达,减少细胞分泌Ⅰ型胶原,对肝星状细胞的活化有明显的抑制作用.     

褪黑素对增生性瘢痕成纤维细胞生物学活性的调控

目的 探讨褪黑素对增生性瘢痕成纤维细胞生物学活性的调控.方法 分离培养增生性瘢痕成纤维细胞,采用四氮唑复合物/硫酸酚嗪甲酯(XTT/PMS)法检测细胞增殖活性,酶联免疫吸附实验(ELISA)检测细胞培养上清液中β1转化生长因子(TGF-β1)含量和荧光定量聚合酶链反应(PCR)检测细胞α平滑肌肌动蛋白(α-SMA)、胶原Ⅰ和胶原ⅢmRNA表达,评价褪黑素和褪黑素受体拮抗剂(luzindole)对增生性瘢痕成纤维细胞生物学活性的影响.结果 褪黑素抑制增生性瘢痕成纤维细胞的增殖,抑制效果呈浓度依赖性(P＜0.05);高浓度褪黑素(10-3 mmol/L)可降低增生性瘢痕成纤维细胞产生TGF-β1 (P＜0.05)和α-SMA mRNA、胶原Ⅰ mRNA的表达(P＜0.05);褪黑素受体拮抗剂能阻断褪黑素对细胞产生TGF-β1和α-SMA、胶原Ⅰ mRNA表达的抑制作用(P＜0.05);但褪黑素对该细胞胶原ⅢmRNA表达无影响(P＞0.05).结论 褪黑素可通过与受体结合调控增生性瘢痕成纤维细胞的生物学活性.     

结缔组织生长因子在关木通相关肾小管间质肾病肾间质纤维化中的作用

目的 通过观察关木通相关肾小管间质肾病(GMT-TIN)肾小管间质内纤维化相关生长因子表达及细胞外基质(ECM)沉积的特点,探讨其纤维化病变发生与发展的有关环节。方法 22例GMT-TIN患者根据病理特点分为急性组8例、慢性轻型组8例和慢性重型组6例,对其纤维化程度进行半定量分析。应用免疫组化SP法观察肾活检组织标本中,肾小管间质内α-平滑肌肌动蛋白(α-SMA)、纤连蛋白(FN)、IV型胶原、结缔组织生长因子(CTGF)和转化生长因子 β1(TGF-β1)的表达情况。分析上述指标之间以及与纤维化病变之间的相关关系。结果 (1)各组肾小管间质内均检测到高表达的α-SMA、FN和IV型胶原,随病变慢性化加重3者表达均明显增强,并与肾间质纤维化程度正相关(P<0.01)。(2)各组肾间质内均出现CTGF及TGF-β1表达,2者间显著正相关(r=O.771,P<0.0001),但在时相上存在一定差异。(3)肾间质内CTGF表达分别与FN及IV型胶原沉积正相关(r=0.6855和0.5964,P<0.01)、与纤维化病变程度正相关(r=0.4941,P<0.05)。结论(1)GMT-TIN肾间质内CTGF的高表达、CTGF与ECM沉积以及纤维化病变的相关性,提示其在GMT-TIN肾间质纤维化病变中起重要作用。(2)CTGF与TGF-β1的相关关系及2者表达时相的差异,进一步支持CTGF可能作为TGF-β1的下游效应因子在纤维化病变     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.