睾酮通过调控趋化因子受体CXCR3影响尿道狭窄疤痕成纤维细胞迁移的实验研究

目的 探索人尿道狭窄疤痕成纤维细胞在不同双氢睾酮(DHT)环境中CXCR3表达水平变化及迁移变化、并探寻涉及的潜在相关生物信号通路。方法 选取不同DHT浓度作用于人尿道狭窄疤痕成纤维细胞,Western-blotting检测不同组成纤维细胞CXCR3受体的表达情况。通过Transwell小室试验研究不同浓度的DHT对成纤维细胞迁移能力的影响。取用不同浓度DHT处理组和酒精刺激后的空白对照组,利用转录组测序技术(RNA-seq)分析寻找DHT与CXCR3潜在相关生物信号通路。结果 Transwell实验证实,随着DHT浓度的逐步升高,成纤维细胞的迁移得以相应减弱。同时阶梯式升高浓度的DHT溶液环境可显著增加各组成纤维细胞CXCR3的表达,加入恩杂鲁胺拮抗雄激素受体(AR)后CXCR3受体表达明显下降。通过RNA测序后发现不同浓度雄激素处理后能显著改变部分基因的表达,主要富集于TGF-β信号通路、细胞黏合信号通路、类风湿炎症信号通路、MAPK信号通路、Ras信号通路、细胞运动骨架信号通路、Hippo信号通路、WNT信号通路等。结论 在高雄激素环境中,人尿道狭窄疤痕成纤维细胞迁移有明显变化,...     

mTOR通路调控肾间质成纤维细胞活化的机制

目的 探讨mTOR信号通路在肾间质成纤维细胞增生活化过程中的调控作用,并研究其抑制剂在抗肾纤维化治疗中的可行性.方法 用8周龄雌性C57BL/6小鼠构建单侧输尿管结扎(UUO)肾间质纤维化动物模型(n=30),按数字随机法分为雷帕霉素组(n=15)及UUO组(n=15).雷帕霉素组术前1d开始腹腔注射雷帕霉素(2 mg·kg-1·d-1)至实验结束;UUO组注射生理盐水.分别于术后1、3、7、14 d处死小鼠(n=3),留肾组织进行相关检测.同时,体外实验评估雷帕霉素对TGF-β诱导鼠成纤维细胞株(NIH3T3细胞)活化的干预作用.结果 UUO小鼠肾组织中活化的肌成纤维细胞[α肌动蛋白(α-SMA)阳性]高表达mTOR通路下游效应因子pS6K.雷帕霉素显著抑制pS6K表达及肾间质中肌成纤维细胞的活化,改善肾小管间质损伤及纤维化程度.实时荧光定量PCR结果提示雷帕霉素组小鼠肾皮质组织中成纤维细胞特异蛋白1 (FSP1)、转化生长因子β(TGF-β)、结缔组织因子(CTGF)及Ⅳ型胶原蛋白基因α1 (Col 4A1)的mRNA水平显著下降.体外实验结果示TGF-β诱导小鼠成纤维细胞株( NIH3T3)的mTOR通路显著活化,并大量合成α-SMA.雷帕霉素能够明显抑制mTOR通路活性,降低细胞的纤维化活性.结论 肾间质纤维化过程中成纤维细胞内的mTOR信号通路高度活化.抑制mTOR通路能够显著降低成纤维细胞的活性,改善肾间质纤维化程度.     

松果菊苷对人增生性瘢痕成纤维细胞TGF-β1/Smads信号通路影响的实验研究

目的 基于TGF-β1/Smads信号通路探讨松果菊苷(echinacoside,ECH)对人增生性瘢痕成纤维细胞(HSFbs)的作用机制.方法 自2019年3月至2020年2月新疆医科大学第一附属医院整形外科体外培养人增生性瘢痕成纤维细胞;通过CCK-8法分析ECH作用24 h和48 h细胞的抑制活性.采用划痕试验检...     

RhoA/Rho激酶信号通路在TGF-β1诱导的人皮肤成纤维细胞表型转化中的作用

目的 观察TGF-[1对人皮肤成纤维细胞表型转化的作用是否受到RhoA/Rho激酶信号通路的影响,以探究该通路是否参与了人皮肤成纤维细胞的表型转化。方法 原代培养人皮肤成纤维细胞,将第4代细胞用TGF-β1( 10 ng/ml)刺激后,分不同作用时间(0、3、6、24h)提取细胞内总蛋白。BCA法测定总蛋白浓度,Western -blot检测α-SMA的表达水平;并用相同的方法检测不同浓度TGF-β1(0、2、10、50 ng/ml)刺激人皮肤成纤维细胞后,α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的表达水平。用RhoA/Rho激酶信号通路阻断剂Y-27632处理后,再用TGF-β1刺激,作为实验组,将未作处理的正常细胞作为空白对照组,Y-27632（10 μmol/L）组作为阴性对照组,只用TGF-β1( 10 ng/ml)刺激组作为阳性对照组,分别检测α-SMA的表达差异。使用ANOVA对蛋白表达量进行统计学分析,P ＜0.05为差异有统计学意义。结果 10 ng/ml TGF-β1按不同作用时间刺激人皮肤成纤维细胞后,α-SMA的表达量分别为：0h为1.0,3h为1.9+0.2、6h为2.1±0.1,24 h为3.1±0.1。24 h较其他3个时间点α-SMA表达量明显上升（n=4,P＜0.05）。不同浓度TGF-β1刺激人皮肤成纤维细胞后,α-SMA的表达量分别为：0 ng/ml为1.0,2 ng/ml为1.4±0.2,10 ng/ml为3.2±0.1,50 ng/ml为3.1±0.2。10 ng/ml表达量明显高于0 ng/ml和2 ng/ml(n =4,P ＜0.05),较50 ng/ml差异无统计学意义(n=4,P＞0.05)。用Y-27632（10 μmol/L）处理后,再用TGF-β1刺激,细胞的表型转化明显受到抑制,实验组α-SMA的表达量(1.2±0.2)较阳性对照组(2.9±0.1)明显降低(n=5,P ＜0.05),与空白对照组(1.0)和阴性对照组(1.1±0.1)相比差异均无统计学意义(n=5,P＞0.05)。结论 RhoA/Rho激酶信号通路参与了 TGF-β1诱导的人皮肤成纤维细胞表型转化过程。     

Yes相关蛋白通过Wnt/β-catenin信号通路调控软骨细胞SOX9表达的实验研究

目的 研究Yes相关蛋白（YAP）通过Wnt/β-catenin信号通路调控软骨细胞SOX9表达的机制。 方法 用siRNA分别沉默YAP基因和LATS1基因来调控YAP的活性,通过Real-time PCR 检测软骨特异性基因和Wnt/β-catenin信号通路下游基因的表达;通过Western Blot检测GSK3β的磷酸化水平、活化β-catenin的蛋白水平以及SOX9的蛋白水平;并通过阿利新蓝染色检测软骨细胞外基质的分泌情况。用siRNA沉默YAP基因后,再用氯化锂干预细胞,通过Western Blot检测SOX9的表达和GSK3β的磷酸化水平。 结果 沉默YAP基因后,磷酸化GSK3β和活化β-catenin的蛋白水平减少,c-Myc和Nanog基因表达减少,SOX9、COL2和Aggrecan基因表达升高,并且软骨细胞分泌基质增多;沉默LATS1基因后,磷酸化GSK3β和活化β-catenin的蛋白水平增加,c-Myc和Nanog基因表达增加,SOX9、COL2和Aggrecan基因表达减少,并且软骨细胞分泌基质减少。此外,氯化锂可以阻断沉默YAP引起的SOX9表达上调。 结论 YAP通过Wnt/β-catenin信号通路调控软骨细胞SOX9的表达。     

Sufu通过抑制Hedgehog信号通路抑制成纤维细胞的纤维化作用

目的 探讨Sufu(suppressor of fused)是否通过抑制Hedgehog信号通路抑制成纤维细胞的纤维化作用.方法 自2019年3—8月通过北部战区总医院烧伤整形科获取手术切除的增生性瘢痕组织和正常皮肤组织,经Western blot检测组织中Sufu的表达情况,原代提取增生性瘢痕成纤维细胞.通过噻唑蓝(...     

TGF-β1对腱鞘成纤维细胞a-SMA及细胞外基质合成的影响

[目的]研究TGF-β1对腱鞘成纤维细胞a-SMA及细胞外基质合成的影响。[方法]培养兔趾深屈肌腱腱鞘成纤维细胞,培养液中加入TGF-β1(5ng/ml)。培养48h后,用Western-Blot检测a-SMA表达;ELISA测定细胞I型胶原及纤维结合素的表达。[结果]TGF-β1(5ng/ml)作用后的成纤维细胞a-SMA表达量明显高于对照组成纤维细胞(P0.05)。TGF-β1同样可以诱导I型胶原及纤维结合素的表达(P0.05)。[结论]TGF-β1能诱导体外培养的腱鞘成纤维细胞I型胶原、纤维结合素及a-SMA的表达,明确了TGF-β1在诱导肌腱愈合后粘连产生的机制,这为肌腱损伤后粘连及瘢痕的预防和治疗在细胞和分子水平提供了依据。     

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。     

结缔组织生长因子影响体外培养瘢痕成纤维细胞ERK/MAPK信号通路活化及增殖的研究

Objective To explore the signal mechanism of proliferation stimulating effect of connec-tive tissue growth factor (CTGF) on hypertrophic scar (HS) derived fibroblasts. Methods <'3>H-TdR in-corporation technique was used to determine the proliferative effect of CTGF at different concentration. Western blot was applied to semi-quantitively analyze the expression of phosphorylated and total ERK1/2 protein after 0, 5, 10, 15, 30, and 60 min of CTGF stimulation, and the relative value of which was de-fined as AI to measure the activation of ERK1/2 signal pathway. PD98059 was admitted to specifically block the ERK1/2 pathway, and subsequently cell proliferation stimulated by CTGF was studied by MTT. Results CTGF could stimulate fibroblasts proliferation with a dose-dependant manner, and activa-ted the ERK1/2 signal pathway, and AI built up to 0.209±0.0201, reaching the apex at 15 min after stimulation performed. Inhibition of ERK1/2 activation by PD98059 suppressed CTGF-mediated HS fi-broblasts proliferation significantly, while OD significantly dropped. Conclusion CTGF induces a prolif-erative response in HS fibroblasts, and this action is mainly dependent on the activation of ERK1/2 signal pathway.     

结缔组织生长因子影响体外培养瘢痕成纤维细胞ERK/MAPK信号通路活化及增殖的研究

Objective To explore the signal mechanism of proliferation stimulating effect of connec-tive tissue growth factor (CTGF) on hypertrophic scar (HS) derived fibroblasts. Methods <'3>H-TdR in-corporation technique was used to determine the proliferative effect of CTGF at different concentration. Western blot was applied to semi-quantitively analyze the expression of phosphorylated and total ERK1/2 protein after 0, 5, 10, 15, 30, and 60 min of CTGF stimulation, and the relative value of which was de-fined as AI to measure the activation of ERK1/2 signal pathway. PD98059 was admitted to specifically block the ERK1/2 pathway, and subsequently cell proliferation stimulated by CTGF was studied by MTT. Results CTGF could stimulate fibroblasts proliferation with a dose-dependant manner, and activa-ted the ERK1/2 signal pathway, and AI built up to 0.209±0.0201, reaching the apex at 15 min after stimulation performed. Inhibition of ERK1/2 activation by PD98059 suppressed CTGF-mediated HS fi-broblasts proliferation significantly, while OD significantly dropped. Conclusion CTGF induces a prolif-erative response in HS fibroblasts, and this action is mainly dependent on the activation of ERK1/2 signal pathway.     

结缔组织生长因子影响体外培养瘢痕成纤维细胞ERK/MAPK信号通路活化及增殖的研究

Objective To explore the signal mechanism of proliferation stimulating effect of connec-tive tissue growth factor (CTGF) on hypertrophic scar (HS) derived fibroblasts. Methods <'3>H-TdR in-corporation technique was used to determine the proliferative effect of CTGF at different concentration. Western blot was applied to semi-quantitively analyze the expression of phosphorylated and total ERK1/2 protein after 0, 5, 10, 15, 30, and 60 min of CTGF stimulation, and the relative value of which was de-fined as AI to measure the activation of ERK1/2 signal pathway. PD98059 was admitted to specifically block the ERK1/2 pathway, and subsequently cell proliferation stimulated by CTGF was studied by MTT. Results CTGF could stimulate fibroblasts proliferation with a dose-dependant manner, and activa-ted the ERK1/2 signal pathway, and AI built up to 0.209±0.0201, reaching the apex at 15 min after stimulation performed. Inhibition of ERK1/2 activation by PD98059 suppressed CTGF-mediated HS fi-broblasts proliferation significantly, while OD significantly dropped. Conclusion CTGF induces a prolif-erative response in HS fibroblasts, and this action is mainly dependent on the activation of ERK1/2 signal pathway.     

结缔组织生长因子影响体外培养瘢痕成纤维细胞ERK/MAPK信号通路活化及增殖的研究

Objective To explore the signal mechanism of proliferation stimulating effect of connec-tive tissue growth factor (CTGF) on hypertrophic scar (HS) derived fibroblasts. Methods <'3>H-TdR in-corporation technique was used to determine the proliferative effect of CTGF at different concentration. Western blot was applied to semi-quantitively analyze the expression of phosphorylated and total ERK1/2 protein after 0, 5, 10, 15, 30, and 60 min of CTGF stimulation, and the relative value of which was de-fined as AI to measure the activation of ERK1/2 signal pathway. PD98059 was admitted to specifically block the ERK1/2 pathway, and subsequently cell proliferation stimulated by CTGF was studied by MTT. Results CTGF could stimulate fibroblasts proliferation with a dose-dependant manner, and activa-ted the ERK1/2 signal pathway, and AI built up to 0.209±0.0201, reaching the apex at 15 min after stimulation performed. Inhibition of ERK1/2 activation by PD98059 suppressed CTGF-mediated HS fi-broblasts proliferation significantly, while OD significantly dropped. Conclusion CTGF induces a prolif-erative response in HS fibroblasts, and this action is mainly dependent on the activation of ERK1/2 signal pathway.     

结缔组织生长因子影响体外培养瘢痕成纤维细胞ERK/MAPK信号通路活化及增殖的研究

Objective To explore the signal mechanism of proliferation stimulating effect of connec-tive tissue growth factor (CTGF) on hypertrophic scar (HS) derived fibroblasts. Methods <'3>H-TdR in-corporation technique was used to determine the proliferative effect of CTGF at different concentration. Western blot was applied to semi-quantitively analyze the expression of phosphorylated and total ERK1/2 protein after 0, 5, 10, 15, 30, and 60 min of CTGF stimulation, and the relative value of which was de-fined as AI to measure the activation of ERK1/2 signal pathway. PD98059 was admitted to specifically block the ERK1/2 pathway, and subsequently cell proliferation stimulated by CTGF was studied by MTT. Results CTGF could stimulate fibroblasts proliferation with a dose-dependant manner, and activa-ted the ERK1/2 signal pathway, and AI built up to 0.209±0.0201, reaching the apex at 15 min after stimulation performed. Inhibition of ERK1/2 activation by PD98059 suppressed CTGF-mediated HS fi-broblasts proliferation significantly, while OD significantly dropped. Conclusion CTGF induces a prolif-erative response in HS fibroblasts, and this action is mainly dependent on the activation of ERK1/2 signal pathway.     

结缔组织生长因子影响体外培养瘢痕成纤维细胞ERK/MAPK信号通路活化及增殖的研究

Objective To explore the signal mechanism of proliferation stimulating effect of connec-tive tissue growth factor (CTGF) on hypertrophic scar (HS) derived fibroblasts. Methods <'3>H-TdR in-corporation technique was used to determine the proliferative effect of CTGF at different concentration. Western blot was applied to semi-quantitively analyze the expression of phosphorylated and total ERK1/2 protein after 0, 5, 10, 15, 30, and 60 min of CTGF stimulation, and the relative value of which was de-fined as AI to measure the activation of ERK1/2 signal pathway. PD98059 was admitted to specifically block the ERK1/2 pathway, and subsequently cell proliferation stimulated by CTGF was studied by MTT. Results CTGF could stimulate fibroblasts proliferation with a dose-dependant manner, and activa-ted the ERK1/2 signal pathway, and AI built up to 0.209±0.0201, reaching the apex at 15 min after stimulation performed. Inhibition of ERK1/2 activation by PD98059 suppressed CTGF-mediated HS fi-broblasts proliferation significantly, while OD significantly dropped. Conclusion CTGF induces a prolif-erative response in HS fibroblasts, and this action is mainly dependent on the activation of ERK1/2 signal pathway.     

结缔组织生长因子影响体外培养瘢痕成纤维细胞ERK/MAPK信号通路活化及增殖的研究

Objective To explore the signal mechanism of proliferation stimulating effect of connec-tive tissue growth factor (CTGF) on hypertrophic scar (HS) derived fibroblasts. Methods <'3>H-TdR in-corporation technique was used to determine the proliferative effect of CTGF at different concentration. Western blot was applied to semi-quantitively analyze the expression of phosphorylated and total ERK1/2 protein after 0, 5, 10, 15, 30, and 60 min of CTGF stimulation, and the relative value of which was de-fined as AI to measure the activation of ERK1/2 signal pathway. PD98059 was admitted to specifically block the ERK1/2 pathway, and subsequently cell proliferation stimulated by CTGF was studied by MTT. Results CTGF could stimulate fibroblasts proliferation with a dose-dependant manner, and activa-ted the ERK1/2 signal pathway, and AI built up to 0.209±0.0201, reaching the apex at 15 min after stimulation performed. Inhibition of ERK1/2 activation by PD98059 suppressed CTGF-mediated HS fi-broblasts proliferation significantly, while OD significantly dropped. Conclusion CTGF induces a prolif-erative response in HS fibroblasts, and this action is mainly dependent on the activation of ERK1/2 signal pathway.     

结缔组织生长因子影响体外培养瘢痕成纤维细胞ERK/MAPK信号通路活化及增殖的研究

Objective To explore the signal mechanism of proliferation stimulating effect of connec-tive tissue growth factor (CTGF) on hypertrophic scar (HS) derived fibroblasts. Methods <'3>H-TdR in-corporation technique was used to determine the proliferative effect of CTGF at different concentration. Western blot was applied to semi-quantitively analyze the expression of phosphorylated and total ERK1/2 protein after 0, 5, 10, 15, 30, and 60 min of CTGF stimulation, and the relative value of which was de-fined as AI to measure the activation of ERK1/2 signal pathway. PD98059 was admitted to specifically block the ERK1/2 pathway, and subsequently cell proliferation stimulated by CTGF was studied by MTT. Results CTGF could stimulate fibroblasts proliferation with a dose-dependant manner, and activa-ted the ERK1/2 signal pathway, and AI built up to 0.209±0.0201, reaching the apex at 15 min after stimulation performed. Inhibition of ERK1/2 activation by PD98059 suppressed CTGF-mediated HS fi-broblasts proliferation significantly, while OD significantly dropped. Conclusion CTGF induces a prolif-erative response in HS fibroblasts, and this action is mainly dependent on the activation of ERK1/2 signal pathway.     

结缔组织生长因子影响体外培养瘢痕成纤维细胞ERK/MAPK信号通路活化及增殖的研究

Objective To explore the signal mechanism of proliferation stimulating effect of connec-tive tissue growth factor (CTGF) on hypertrophic scar (HS) derived fibroblasts. Methods <'3>H-TdR in-corporation technique was used to determine the proliferative effect of CTGF at different concentration. Western blot was applied to semi-quantitively analyze the expression of phosphorylated and total ERK1/2 protein after 0, 5, 10, 15, 30, and 60 min of CTGF stimulation, and the relative value of which was de-fined as AI to measure the activation of ERK1/2 signal pathway. PD98059 was admitted to specifically block the ERK1/2 pathway, and subsequently cell proliferation stimulated by CTGF was studied by MTT. Results CTGF could stimulate fibroblasts proliferation with a dose-dependant manner, and activa-ted the ERK1/2 signal pathway, and AI built up to 0.209±0.0201, reaching the apex at 15 min after stimulation performed. Inhibition of ERK1/2 activation by PD98059 suppressed CTGF-mediated HS fi-broblasts proliferation significantly, while OD significantly dropped. Conclusion CTGF induces a prolif-erative response in HS fibroblasts, and this action is mainly dependent on the activation of ERK1/2 signal pathway.     

结缔组织生长因子影响体外培养瘢痕成纤维细胞ERK/MAPK信号通路活化及增殖的研究

Objective To explore the signal mechanism of proliferation stimulating effect of connec-tive tissue growth factor (CTGF) on hypertrophic scar (HS) derived fibroblasts. Methods <'3>H-TdR in-corporation technique was used to determine the proliferative effect of CTGF at different concentration. Western blot was applied to semi-quantitively analyze the expression of phosphorylated and total ERK1/2 protein after 0, 5, 10, 15, 30, and 60 min of CTGF stimulation, and the relative value of which was de-fined as AI to measure the activation of ERK1/2 signal pathway. PD98059 was admitted to specifically block the ERK1/2 pathway, and subsequently cell proliferation stimulated by CTGF was studied by MTT. Results CTGF could stimulate fibroblasts proliferation with a dose-dependant manner, and activa-ted the ERK1/2 signal pathway, and AI built up to 0.209±0.0201, reaching the apex at 15 min after stimulation performed. Inhibition of ERK1/2 activation by PD98059 suppressed CTGF-mediated HS fi-broblasts proliferation significantly, while OD significantly dropped. Conclusion CTGF induces a prolif-erative response in HS fibroblasts, and this action is mainly dependent on the activation of ERK1/2 signal pathway.     

成纤维细胞在泌尿系疾病病因机制及治疗中的研究进展

成纤维细胞的增生、变性及分泌是诸多泌尿系统疾病的发病机制之一, 如肾脏纤维化、输尿管狭窄、膀胱颈挛缩、尿道狭窄、阴茎硬化性苔藓样变以及阴茎硬结症等。通过调控与成纤维细胞增殖及分化等相关的病因机制, 可达到控制及治愈疾病的目的。本文就成纤维细胞的生物学特征及其在泌尿系统疾病机制中的发生发展和相关治疗的研究进展作一综述。     

不同亚群成纤维细胞及细胞外基质在增生性瘢痕形成中的作用

增生性瘢痕是创面愈合后成纤维细胞异常增殖和胶原过度沉积的结果。但并非所有的皮肤成纤维细胞都参与介导了增生性瘢痕的形成,不同亚群或位于真皮不同层次的成纤维细胞发挥着不同的功能。此外,细胞外基质亦对皮肤纤维化产生了重要影响。因此,阐明上述因素与增生性瘢痕形成的关系可能有助于增生性瘢痕的防治。