转化生长因子β_1对人巩膜成纤维细胞Smad泛素化调节因子2和Ⅰ型胶原α_1影响的研究
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摘要
目的探讨Smad泛素化调节因子2(Smurf2)在人胚胎眼巩膜成纤维细胞(HFSFs)中的表达,检测转化生长因子β_1(TGF-β_1)影响下HFSFs中Smurf2和Ⅰ型胶原α_1(COLIA1)含量的变化。方法 HFSFs复苏并稳定传代后,用免疫细胞化学法检测细胞中Smurf2的表达。分别用不同浓度的TGF-β_1(0、1 ng/ml、5 ng/ml及10 ng/ml)处理HFSFs 24 h以及10 ng/ml TGF-β_1处理HFSFs不同时长(1 h、6 h、12 h及24 h)后,用实时荧光定量法检测各组Smurf2信使核糖核酸(mRNA)和COLIA1 mRNA表达水平。数据以均数±标准差(±s)描述,不同浓度和不同时长的组间比较采用单因素方差分析,当差异有统计学意义时,进一步采用SNK法两两比较。结果免疫细胞化学检测的结果显示,HFSFs中有COLIA1 mRNA和Smurf2蛋白的表达。与空白对照组比较,TGF-β_110 ng/ml组COLIA1 mRNA的表达呈上升趋势(F=3. 17,P <0. 05),TGF-β_15 ng/ml、10 ng/ml组Smurf2 mRNA表达上调(F=2. 35,2. 00; P <0. 05)。10 ng/ml TGF-β_1干预HFSFs培养1 h、6 h、12 h及24 h后,24 h组COLIA1 mRNA表达显著升高(F=29. 20,P <0. 05)。Smurf2 mRNA表达改变呈先上升后下降趋势,在6 h达到最高(F=10. 65,P <0. 05)。结论 TGF-β_1可能通过调控HFSFs Smurf2的表达,诱导HFSFs COLIA1的合成。
Objective The aim of this study was to investigate the expression of Smad ubiquitination regulatory factor 2 (Smurf 2) in human fetal scleral fibroblasts (HFSFs) cells,and the changes of Smurf 2 and collagen type Ⅰ α_1 (COLIA1) in HFSFs induced by transforming growth factor-beta1 (TGF-β_1). Methods After recovery and stable passage of HFSFs,expression of Smurf2 in cells was detected by immunocytochemistry. HFSFs were treated with different concentrations of TGF-β_1 (0,1 ng/ml,5 ng/ml,10 ng/ml) for 24 hours,respectively,and co-cultured with TGF-β_1 (10 ng/ml) for 1 hour,6 hours,12 hours,and 24 hours. Then,the expression of COLIA1 messenger RNA (mRNA) and Smurf2 mRNA were detected by reverse transcription-polymerase chain reaction. Data was described by (± s).One-way ANOVA was used for comparison between groups,then SNK test was used if difference was statistically significant. Results The results showed that COLIA1 mRNA and Smurf2 protein expressed in HFSFs. COLIA1 mRNA expressed an upward trend in 10 ng/ml TGF-β_1 group compared with control group (F = 3. 17,P < 0. 05),as well as Smurf2 mRNA in 5 ng/ml TGF-β_1 group and 10 ng/ml TGF-β_1 group (F= 2. 35,2. 00; P < 0. 05). After 1 hour,6 hours,12 hours,24 hours for HFSFs co-cultured with 10 ng/ml TGF-β_1,the expression of COLIA1 mRNA increased significantly in 24 hours group (F = 29. 20,P <0. 05). While,the expression of Smurf2 mRNA increased first and then decreased,reaching the highest level at 6 hours (F = 10. 65,P < 0. 05). Conclusion TGF-β_1 may induce the synthesis of HFSFs COLIA1 by regulating the expression of HFSFs Smurf2.
Objective The aim of this study was to investigate the expression of Smad ubiquitination regulatory factor 2 (Smurf 2) in human fetal scleral fibroblasts (HFSFs) cells,and the changes of Smurf 2 and collagen type Ⅰ α_1 (COLIA1) in HFSFs induced by transforming growth factor-beta1 (TGF-β_1). Methods After recovery and stable passage of HFSFs,expression of Smurf2 in cells was detected by immunocytochemistry. HFSFs were treated with different concentrations of TGF-β_1 (0,1 ng/ml,5 ng/ml,10 ng/ml) for 24 hours,respectively,and co-cultured with TGF-β_1 (10 ng/ml) for 1 hour,6 hours,12 hours,and 24 hours. Then,the expression of COLIA1 messenger RNA (mRNA) and Smurf2 mRNA were detected by reverse transcription-polymerase chain reaction. Data was described by (± s).One-way ANOVA was used for comparison between groups,then SNK test was used if difference was statistically significant. Results The results showed that COLIA1 mRNA and Smurf2 protein expressed in HFSFs. COLIA1 mRNA expressed an upward trend in 10 ng/ml TGF-β_1 group compared with control group (F = 3. 17,P < 0. 05),as well as Smurf2 mRNA in 5 ng/ml TGF-β_1 group and 10 ng/ml TGF-β_1 group (F= 2. 35,2. 00; P < 0. 05). After 1 hour,6 hours,12 hours,24 hours for HFSFs co-cultured with 10 ng/ml TGF-β_1,the expression of COLIA1 mRNA increased significantly in 24 hours group (F = 29. 20,P <0. 05). While,the expression of Smurf2 mRNA increased first and then decreased,reaching the highest level at 6 hours (F = 10. 65,P < 0. 05). Conclusion TGF-β_1 may induce the synthesis of HFSFs COLIA1 by regulating the expression of HFSFs Smurf2.
引文
[1]李翯,周翔天,瞿佳.碱性成纤维细胞生长因子和转化生长因子-β在实验性近视中的研究进展[J].眼视光学杂志,2004,6(3):193-195,198.
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[3]杨俊侠,曹述任,张敏.Smad泛素化调节因子2在转化生长因子β1诱导人肺成纤维细胞活化中的作用及其分子机制[J].吉林大学学报(医学版),2015,41(5):891-897.
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[8]Mc Brien NA,Jobling AI,Gentle A.Biomechanics of the sclera in myopia:extracellular and cellular factors[J].Optom Vis Sci,2009,86(1):E23-E30.
[9]Lin HJ,Wan L,Tsai Y,et al.Sclera-related gene polymorphisms in high myopia[J].Mol Vis,2009,15(176-178):1655-1663.
[10]Kusakari T,Sato T,Tokoro T.Visual deprivation stimulates the exchange of the fibrous sclera into the cartilaginous sclera in chicks[J].Exp Eye Res,2001,73(4):533-546.
[11]Jobling AI,Gentle A,Metlapally R,et al.Regulation of scleral cell contraction by transforming growth factor-beta and stress:competing roles in myopic eye growth[J].J Biol Chem,2009,284(4):2072-2079.
[12]Mcbrien NA,Jobling AI,Gentle A.Biomechanics of the sclera in myopia:extracellular and cellular factors[J].Optom Vis Sci,2009,86(1):E23-E30.
[13]Seko Y,Shimokawa H,Tokoro T.Expression of bFGF and TGF-beta 2 in experimental myopia in chicks[J].Invest Ophthalmol Vis Sci,1995,36(6):1183-1187.
[14]Honda S,Fujii S,Sekiya Y,et al.Retinal control on the axial length mediated by transforming growth factor-beta in chick eye[J].Invest Ophthalmol Vis Sci,1996,37(12):2519-2526.
[15]曾爱萍,曾水清,程扬.转化生长因子β1对培养的人RPE细胞MMP和TIMP-1 mRNA表达的影响[J].眼科新进展,2006,26(2):81-84.
[16]Xiaofen Z,Renyuan C.Effects of TGF-βon human embryonic retinal pigment epithelium cells[J].Chinese Ophthalmic Research,2007,25(1):14-17.
[17]Siegwart J,Norton TT.Selective regulation of MMP and TIMPmRNA levels in tree shrew sclera during minus lens compensation and recovery[J].Invest Ophthalmol Vis Sci,2005,46(10):3484-3492.
[18]Liu R,Ahmed KM,Nantajit D,et al.Therapeutic effects ofα-lipoic acid on bleomycin-induced pulmonary fibrosis in rats[J].Int J Mol Med,2007,19(6):865-873.
[19]刘海霞,杨坤禹,项楠,等.转化生长因子-β1转基因小鼠巩膜厚度研究[J].华中科技大学学报(医学版),2007,36(5):655-657,670.
[20]Edwards DR,Murphy G,Reynolds JJ,et al.Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor[J].EMBO J,1987,6(7):1899-1904.
[21]Hu J,Cui D,Yang X,et al.Bone morphogenetic protein-2:a potential regulator in scleral remodeling[J].Mol Vis,2008,14(12):2373-2380.
[22]邢凯,吴宁玲,亢泽峰,等.高度近视眼巩膜细胞外基质中相关胶原分子机制的研究进展[J].中华眼科医学杂志(电子版),2018,8(1):44-48.
[23]Shelton L,Rada JS.Effects of cyclic mechanical stretch on extracellular matrix synthesis by human scleral fibroblasts[J].Exp Eye Res,2007,84(2):314-322.
[24]蔡瑜,徐奕,沈锡中.Smad泛素化调节因子2在肝纤维化过程中对结缔组织生长因子的作用研究[J].内科理论与实践,2012,7(4):305-309.
[25]Inoue Y,Imamura T.Regulation of TGF-beta family signaling by E3 ubiquitin ligases[J].Cancer Sci,2008,99(11):2107-2112.
[26]Izzi L,Attisano L.Regulation of the TGFbeta signalling pathway by ubiquitin-mediated degradation[J].Oncogene,2004,23(11):2071-2078.
[27]Itoh S,ten Dijke P.Negative regulation of TGF-beta receptor/Smad signal transduction[J].Curr Opin Cell Biol,2007,19(2):176-184.
[28]Ito I,Hanyu A,Wayama M,et al.Estrogen inhibits transforming growth factor beta signaling by promoting Smad2/3 degradation[J].J Biol Chem,2010,285(19):14747-14755.
[29]Bizet AA,Tran-Khanh N,Saksena A,et al.CD109-mediated degradation of TGF-βreceptors and inhibition of TGF-βresponses involve regulation of SMAD7 and Smurf2 localization and function[J].J Cell Biochem,2012,113(1):238-246.
[30]Yang Q,Chen SP,Zhang XP,et al.Smurf2 participates in human trophoblast cell invasion by inhibiting TGF-beta type Ireceptor[J].J Histochem Cytochem,2009,57(6):605-612.
[31]Chong PA,Lin H,Wrana JL,et al.An expanded WW domain recognition motif revealed by the interaction between Smad7 and the E3 ubiquitin ligase Smurf2[J].J Biol Chem,2006,281(25):17069-17075.
[32]Morén A,Imamura T,Miyazono K,et al.Degradation of the tumor suppressor Smad4 by WW and HECT domain ubiquitin ligases[J].J Biol Chem,2005,280(23):22115-22123.
[33]Nomura N,Sasamoto S,Ishii S,et al.Isolation of human c DNAclones of ski and the ski-related gene,sno[J].Nucleic Acids Res,1989,17(14):5489-5500.
[34]DebigaréR,Price SR.Proteolysis,the ubiquitin-proteasome system,and renal diseases[J].Am J Physiol Renal Physiol,2003,285(1):F1-F8.
[35]Asano Y,Ihn H,Yamane K,et al.Impaired Smad7-Smurfmediated negative regulation of TGF-beta signaling in scleroderma fibroblasts[J].J Clin Invest,2004,113(2):253-264.
[36]刘晓政,田春阳,夏永欣.Smurf在肝纤维化中的表达及其对TGF-β/Smad信号传导的影响[J].广东医学,2017,38(6):834-838.
[37]Ohashi N,Yamamoto T,Uchida C,et al.Transcriptional induction of Smurf2 ubiquitin ligase by TGF-beta[J].FEBSLett,2005,579(12):2557-2563.
[38]潘红卫,高岩,曾骏文.转化生长因子β1对小鼠巩膜成纤维细胞DNA和胶原合成的影响及其在近视形成中的作用[J].中国临床康复,2005,9(18):160-162.
[39]Li H,Cui D,Zhao F,et al.BMP-2 is involved in scleral remodeling in myopia development[J].PLo S One,2015,10(5):e0125219.
[2]Praveen K,Gal LC,Michael B.Smurfs in protein homeostasis,signaling,and cancer[J].Front Oncol,2018,8:295.
[3]杨俊侠,曹述任,张敏.Smad泛素化调节因子2在转化生长因子β1诱导人肺成纤维细胞活化中的作用及其分子机制[J].吉林大学学报(医学版),2015,41(5):891-897.
[4]Fukasawa H,Yamamoto T,Togawa A,et al.Ubiquitindependent degradation of SnoN and Ski is increased in renal fibrosis induced by obstructive injury[J].Kidney Int,2006,69(10):1733-1740.
[5]Cai Y,Shen XZ,Zhou CH,et al.Abnormal expression of Smurf2 during the process of rat liver fibrosis[J].Chin J Dig Dis,2006,7(4):237-245.
[6]Mc Brien NA,Gentle A.Role of the sclera in the development and pathological complications of myopia[J].Prog Retin Eye Res,2003,22(3):307-338.
[7]Norton TT,Rada JA.Reduced extracellular matrix in mammalian sclera with induced myopia[J].Vision Res,1995,35(9):1271-1281.
[8]Mc Brien NA,Jobling AI,Gentle A.Biomechanics of the sclera in myopia:extracellular and cellular factors[J].Optom Vis Sci,2009,86(1):E23-E30.
[9]Lin HJ,Wan L,Tsai Y,et al.Sclera-related gene polymorphisms in high myopia[J].Mol Vis,2009,15(176-178):1655-1663.
[10]Kusakari T,Sato T,Tokoro T.Visual deprivation stimulates the exchange of the fibrous sclera into the cartilaginous sclera in chicks[J].Exp Eye Res,2001,73(4):533-546.
[11]Jobling AI,Gentle A,Metlapally R,et al.Regulation of scleral cell contraction by transforming growth factor-beta and stress:competing roles in myopic eye growth[J].J Biol Chem,2009,284(4):2072-2079.
[12]Mcbrien NA,Jobling AI,Gentle A.Biomechanics of the sclera in myopia:extracellular and cellular factors[J].Optom Vis Sci,2009,86(1):E23-E30.
[13]Seko Y,Shimokawa H,Tokoro T.Expression of bFGF and TGF-beta 2 in experimental myopia in chicks[J].Invest Ophthalmol Vis Sci,1995,36(6):1183-1187.
[14]Honda S,Fujii S,Sekiya Y,et al.Retinal control on the axial length mediated by transforming growth factor-beta in chick eye[J].Invest Ophthalmol Vis Sci,1996,37(12):2519-2526.
[15]曾爱萍,曾水清,程扬.转化生长因子β1对培养的人RPE细胞MMP和TIMP-1 mRNA表达的影响[J].眼科新进展,2006,26(2):81-84.
[16]Xiaofen Z,Renyuan C.Effects of TGF-βon human embryonic retinal pigment epithelium cells[J].Chinese Ophthalmic Research,2007,25(1):14-17.
[17]Siegwart J,Norton TT.Selective regulation of MMP and TIMPmRNA levels in tree shrew sclera during minus lens compensation and recovery[J].Invest Ophthalmol Vis Sci,2005,46(10):3484-3492.
[18]Liu R,Ahmed KM,Nantajit D,et al.Therapeutic effects ofα-lipoic acid on bleomycin-induced pulmonary fibrosis in rats[J].Int J Mol Med,2007,19(6):865-873.
[19]刘海霞,杨坤禹,项楠,等.转化生长因子-β1转基因小鼠巩膜厚度研究[J].华中科技大学学报(医学版),2007,36(5):655-657,670.
[20]Edwards DR,Murphy G,Reynolds JJ,et al.Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor[J].EMBO J,1987,6(7):1899-1904.
[21]Hu J,Cui D,Yang X,et al.Bone morphogenetic protein-2:a potential regulator in scleral remodeling[J].Mol Vis,2008,14(12):2373-2380.
[22]邢凯,吴宁玲,亢泽峰,等.高度近视眼巩膜细胞外基质中相关胶原分子机制的研究进展[J].中华眼科医学杂志(电子版),2018,8(1):44-48.
[23]Shelton L,Rada JS.Effects of cyclic mechanical stretch on extracellular matrix synthesis by human scleral fibroblasts[J].Exp Eye Res,2007,84(2):314-322.
[24]蔡瑜,徐奕,沈锡中.Smad泛素化调节因子2在肝纤维化过程中对结缔组织生长因子的作用研究[J].内科理论与实践,2012,7(4):305-309.
[25]Inoue Y,Imamura T.Regulation of TGF-beta family signaling by E3 ubiquitin ligases[J].Cancer Sci,2008,99(11):2107-2112.
[26]Izzi L,Attisano L.Regulation of the TGFbeta signalling pathway by ubiquitin-mediated degradation[J].Oncogene,2004,23(11):2071-2078.
[27]Itoh S,ten Dijke P.Negative regulation of TGF-beta receptor/Smad signal transduction[J].Curr Opin Cell Biol,2007,19(2):176-184.
[28]Ito I,Hanyu A,Wayama M,et al.Estrogen inhibits transforming growth factor beta signaling by promoting Smad2/3 degradation[J].J Biol Chem,2010,285(19):14747-14755.
[29]Bizet AA,Tran-Khanh N,Saksena A,et al.CD109-mediated degradation of TGF-βreceptors and inhibition of TGF-βresponses involve regulation of SMAD7 and Smurf2 localization and function[J].J Cell Biochem,2012,113(1):238-246.
[30]Yang Q,Chen SP,Zhang XP,et al.Smurf2 participates in human trophoblast cell invasion by inhibiting TGF-beta type Ireceptor[J].J Histochem Cytochem,2009,57(6):605-612.
[31]Chong PA,Lin H,Wrana JL,et al.An expanded WW domain recognition motif revealed by the interaction between Smad7 and the E3 ubiquitin ligase Smurf2[J].J Biol Chem,2006,281(25):17069-17075.
[32]Morén A,Imamura T,Miyazono K,et al.Degradation of the tumor suppressor Smad4 by WW and HECT domain ubiquitin ligases[J].J Biol Chem,2005,280(23):22115-22123.
[33]Nomura N,Sasamoto S,Ishii S,et al.Isolation of human c DNAclones of ski and the ski-related gene,sno[J].Nucleic Acids Res,1989,17(14):5489-5500.
[34]DebigaréR,Price SR.Proteolysis,the ubiquitin-proteasome system,and renal diseases[J].Am J Physiol Renal Physiol,2003,285(1):F1-F8.
[35]Asano Y,Ihn H,Yamane K,et al.Impaired Smad7-Smurfmediated negative regulation of TGF-beta signaling in scleroderma fibroblasts[J].J Clin Invest,2004,113(2):253-264.
[36]刘晓政,田春阳,夏永欣.Smurf在肝纤维化中的表达及其对TGF-β/Smad信号传导的影响[J].广东医学,2017,38(6):834-838.
[37]Ohashi N,Yamamoto T,Uchida C,et al.Transcriptional induction of Smurf2 ubiquitin ligase by TGF-beta[J].FEBSLett,2005,579(12):2557-2563.
[38]潘红卫,高岩,曾骏文.转化生长因子β1对小鼠巩膜成纤维细胞DNA和胶原合成的影响及其在近视形成中的作用[J].中国临床康复,2005,9(18):160-162.
[39]Li H,Cui D,Zhao F,et al.BMP-2 is involved in scleral remodeling in myopia development[J].PLo S One,2015,10(5):e0125219.