【作者】 陈丽娟；

【导师】 胡建民；

【作者基本信息】 福建医科大学 ， 眼科学， 2018， 硕士

【摘要】 目的TGF-β1/Smad信号通路是巩膜重塑的重要信号通路之一,Smurf2(Smad ubiquitination regulatory factor)是TGF-β1/Smad信号通路中的枢纽蛋白。本研究探讨Smurf2在人胚胎眼巩膜成纤维细胞(Human fetal scleral fibroblasts,HFSFs细胞)中表达,及TGF-β1作用下HFSFs细胞中Smurf2和效应蛋白COL1A1含量的变化,以期阐明TGF-β1对HFSFs细胞Smurf2通路的作用。方法HFSFs细胞复苏并稳定传代后,用免疫细胞化学法检测细胞中Smurf2蛋白的表达;分别用不同浓度TGF-β1(0ng/ml、1ng/ml、5ng/ml、10ng/ml)处理HFSF细胞24h以及10ng/ml TGF-β1处理HFSFs细胞不同时长(1h、6h、12h、24h)后,用实时荧光定量法检测各组Smurf2 mRNA和COL1A1 mRNA表达水平;用蛋白免疫印迹(Western-Blot)法检测各组Smurf蛋白2和COL1A1蛋白的表达水平。结果1.Smurf2在HFSFs细胞的表达:免疫细胞化学检测的结果显示HFSFs细胞中有Smurf2蛋白的表达;2.不同浓度TGF-β1干预对HFSFs细胞Smurf2和CoL1A1表达的影响:5 ng/ml、10 ng/mlTGF-β1干预组Smurf2 mRNA表达上调(P=0.002、0.028)。1ng/ml和5 ng/mlTGF-β1干预组Smurf2蛋白表达上调(P=0.011、0.037)。其中,10 ng/ml TGF-β1干预组COL1A1 mRNA和蛋白表达显著升高(P<0.01)。3.不同时长TGF-β1干预HFSFs细胞Smurf2和CoL1A1表达的影响:10 ng/ml TGF-β1共培养HFSFs细胞1h、6h、12h、24h后,COL1A1 mRNA 24h干预组表达显著升高(P=0.033);COL1A1蛋白表达改变呈先增加后降低趋势,6h组COL1A1蛋白表达显著升高(P=0.003),12h表达呈下降趋势(P=0.121),24h组表达有所回升(P=0.017)。Smurf2 mRNA和蛋白表达改变呈先增加后降低趋势,6h组Smurf2 mRNA和蛋白的表达升到最高(P=0.024、0.044);而在24h下降至与未刺激无显著差异(P=0.750,>0.05)。结论1.Smurf2蛋白在HFSFs细胞中有表达。2.10ng/mlTGF-β1干预早期促进Smurf2mRNA和蛋白的表达,可有效诱导HFSFs细胞I型胶原的合成。3.Smurf2蛋白可能参与调控HFSFs细胞中TGF-β1/Smad信号通路的调控。更多还原

【Abstract】 Objective TGF-β1/Smad signaling pathway is one of the important signaling pathways in scleral remodeling,and Smurf2 plays a pivotal role in TGF-β1/Smad signaling pathway.In this experiment,the expression of Smurf2 in HFSFs cells and the changes of Smurf2 and COL1A1 levels in HFSFs cells treated with TGF-β1 were investigated.Methods Recovery of HFSFs cells and then passage them steadily,Smurf2 protein was detected by immunohistochemistry;HFSFs cells were treated with different concentrations of TGF-β1(0 ng/ml,1 ng/ml,5 ng/ml,10 ng/ml)for 24 h;HFSFs cells were co-cultured with TGF-β1(10ng/ml)for 1h,6h,12 h and 24 h.Then,the expression levels of COL1A1 m RNA and Smurf2 m RNA were detected by real-time fluorescence quantification and protein levels were detected by Western blot(Western-Blot)method.Results 1.Smurf2 protein expressed in HFSFs assayed by Immunohistochemistry: The positive expression of Smurf2 protein in HFSFs cells was observed by confocal microscopy.2.Effects of different concentrations of TGF-beta 1 on HFSFs cells The expression of Smurf2 m RNA was up-regulated in the 5 ng/ml and 10 ng/ml TGF-β1 intervention groups((P=0.002、0.028)).Smurf2 protein expression was increased in the 1 ng/ml and 5 ng/ml TGF-β1 intervention groups(P=0.011、0.037).The expression of COL1A1 m RNA and protein was significantly increased in the 10 ng/ml TGF-β1 intervention group(P<0.01).3.The effect of TGF-beta 1 on the level of Smurf2 and COL1A1 in HFSFs cell: Co-cultured HFSFs cells with 10ng/ml TGF-beta 1 for 1h,6h,12 h and 24 h,the expression of COL1A1 mRNA was significantly increased in Group 24h(P=0.033)。The expression of COL1A1 protein was significantly increased in Group 6h(P=0.003).The expression of COL1A1 protein was decreased at 12 h(P=0.121).Group 24 h return to a level similar to basal expression(P=0.017).The expression of Smurf2 m RNA and protein increased firstly and then decreased.The highest level of Smurf2 m RNA and protein was observed at 6h after co-culture(P=0.024、0.044).At 24 h,the level of Smurf2 m RNA showed no difference when compared with 0h(P=0.750,>0.05).Conclusions 1.Smurf2 protein is expressed in HFSFs cells.2.The early stage of intervention of ten ng/ml TGF-β1 promotes the expression of Smurf2 m RNA and protein,which can effectively induce the synthesis of type I collagen in HFSFs cells.3.Smurf2 protein may be involved in regulating HFSFs TGF-beta 1 / Smad signaling pathways in cells of regulation.更多还原