猪脱细胞真皮基质对小鼠创面毛囊再生中基质细胞衍生因子-1及Wnt3a/β-catenin信号通路表达的影响
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摘要
目的探讨猪脱细胞真皮基质(ADM)对小鼠创面毛囊再生中基质细胞衍生因子-1(SDF-1)及Wnt3a/β-catenin信号通路表达的影响。方法取新鲜猪皮,经脱细胞处理后制成微粒状,灭菌,密封,常温保存,以备后面实验使用。制作18只C57BL/6小鼠背部全层皮肤缺损模型,以脊柱为中线,在左右各制作直径为6 mm的缺损,左右侧分别以纱布和猪ADM覆盖,隔天纱布侧换药,猪ADM侧不做处理,于模型建立第7天,按照所用材料不同分为纱布组与猪ADM组,再按所取部位不同分为纱布窗口组、猪ADM窗口组、纱布创面组、猪ADM创面组、纱布创缘组、猪ADM创缘组,其中9只小鼠组织用于蛋白质印迹法检测,另外9只小鼠组织用于免疫组织化学检测。通过蛋白质印迹法检测肿瘤坏死因子-α(TNF-α)、β-catenin、转化生长因子-β(TGF-β)、Wnt3a、血管内皮生长因子(VEGF)、SDF-1、成纤维细胞生长因子(FGF) 2、FGF9、AKT的蛋白表达,免疫组织化学检测Wnt3a、SDF-1、FGF2、FGF9的表达。数据比较采用独立样本t检验。结果在小鼠模型建立第7天创面中,蛋白质印迹法检测β-catenin、Wnt3a、SDF-1的蛋白表达量均是猪ADM窗口组(0. 533±0. 058、0. 446±0. 039、0. 972±0. 048)高于纱布窗口组(0. 401±0. 005、0. 132±0. 022、0. 175±0. 036),差异均有统计学意义(t=3. 996、12. 230、23. 130,P值均小于0. 05)。β-catenin、Wnt3a、SDF-1的蛋白表达量均是猪ADM创面组(0. 557±0. 009、0. 626±0. 066、0. 868±0. 102)高于纱布创面组(0. 302±0. 010、0. 109±0. 019、0. 036±0. 009),差异均有统计学意义(t=32. 830、13. 020、14. 130,P值均小于0. 05)。Wnt3a、SDF-1的蛋白表达量均是猪ADM创缘组(0. 419±0. 014、0. 370±0. 069)高于纱布创缘组(0. 115±0. 020、0. 056±0. 007),差异均有统计学意义(t=21. 460、7. 825,P值均小于0. 05)。免疫组织化学检测结果显示Wnt3a、SDF-1、FGF2、FGF9在猪ADM组中的表达高于纱布组,且阳性细胞主要分布于毛囊细胞周围。结论猪ADM在小鼠创面中可能通过上调SDF-1及Wnt3a/β-catenin信号通路的表达而促进毛囊的再生。
Objective To investigate the effects of porcine acellular dermal matrix( ADM) on the expression of stromal cell-derived factor-1( SDF-1) and Wnt3 a/β-catenin signaling pathway in mouse hair follicle regeneration. Methods Fresh pig skin was taken for dermal matrix,and after decellularization treatment was sterilized,sealed,and stored at room temperature for later use. Eighteen full-thickness skin defect models of C57 BL/6 mice were made with the spine as the midline,6 mm diameter defects were made on the left and right sides. Left and right sides were covered with gauze and porcine ADM,respectively. The gauze side dressing changed the next day,but the porcine ADM side did nothing. On the 7 th day after the model was established,according to different materials,it was divided into gauze group and porcine ADM group,and then according to the different parts taken divided into gauze window group,porcine ADM window group,gauze wound group,porcine ADM wound group,gauze wound edge group and porcine ADM wound edge group. Tissue of nine mice were taken for Western blotting test protein expressions of tumor necrosis factor-α( TNF-α), β-catenin, transforming growth factor-β( TGF-β), Wnt3 a, vascular endothelial growth factor( VEGF),stromalcell derived factor-1( SDF-1),fibroblast growth factor2( FGF2),fibroblast growth factor9( FGF9),AKT. The others were taken for immunohistochemistry for detection of Wnt3 a,SDF-1,FGF2,FGF9. Data comparisons were performed using independent sample t test. Results In the 7 th day wound of mice,Western blotting showed that the protein expression levels of β-catenin,Wnt3 a,SDF-1 were higher in porcine ADM window group( 0. 533 ± 0. 058,0. 446 ± 0. 039,0. 972 ±0. 048) than the gauze window group( 0. 401 ± 0. 005,0. 132 ± 0. 022,0. 175 ± 0. 036),and the differences were statistically significant( t = 3. 996,12. 230,23. 130; with P values below 0. 05). The protein expression levels of β-catenin,Wnt3 a,SDF-1 were higher in the porcine ADM wound group( 0. 557 ±0. 009,0. 626 ± 0. 066,0. 868 ± 0. 102) than in the gauze wound group( 0. 302 ± 0. 010,0. 109 ± 0. 019,0. 036 ± 0. 009),and the differences were statistically significant( t = 32. 830,13. 020,14. 130; with P values below 0. 05). And the protein expression levels of Wnt3 a,SDF-1 in the porcine ADM wound margin group( 0. 419 ± 0. 014,0. 370 ± 0. 069) were higher than those in the gauze margin group( 0. 115 ± 0. 020,0. 056 ± 0. 007),and the differences were statistically significant( t = 21. 460,7. 825; with P values below0. 05). Immunohistochemistry results showed that Wnt3 a,SDF-1,FGF2 and FGF9 in the porcine ADM group were higher than those in the gauze group,and the positive cells were mainly distributed around hair follicle cells. Conclusion In the wound repair process,porcine ADM may promotes the regeneration of hair follicles by up-regulating the expression of SDF-1 and Wnt3 a/β-catenin signaling pathway.
Objective To investigate the effects of porcine acellular dermal matrix( ADM) on the expression of stromal cell-derived factor-1( SDF-1) and Wnt3 a/β-catenin signaling pathway in mouse hair follicle regeneration. Methods Fresh pig skin was taken for dermal matrix,and after decellularization treatment was sterilized,sealed,and stored at room temperature for later use. Eighteen full-thickness skin defect models of C57 BL/6 mice were made with the spine as the midline,6 mm diameter defects were made on the left and right sides. Left and right sides were covered with gauze and porcine ADM,respectively. The gauze side dressing changed the next day,but the porcine ADM side did nothing. On the 7 th day after the model was established,according to different materials,it was divided into gauze group and porcine ADM group,and then according to the different parts taken divided into gauze window group,porcine ADM window group,gauze wound group,porcine ADM wound group,gauze wound edge group and porcine ADM wound edge group. Tissue of nine mice were taken for Western blotting test protein expressions of tumor necrosis factor-α( TNF-α), β-catenin, transforming growth factor-β( TGF-β), Wnt3 a, vascular endothelial growth factor( VEGF),stromalcell derived factor-1( SDF-1),fibroblast growth factor2( FGF2),fibroblast growth factor9( FGF9),AKT. The others were taken for immunohistochemistry for detection of Wnt3 a,SDF-1,FGF2,FGF9. Data comparisons were performed using independent sample t test. Results In the 7 th day wound of mice,Western blotting showed that the protein expression levels of β-catenin,Wnt3 a,SDF-1 were higher in porcine ADM window group( 0. 533 ± 0. 058,0. 446 ± 0. 039,0. 972 ±0. 048) than the gauze window group( 0. 401 ± 0. 005,0. 132 ± 0. 022,0. 175 ± 0. 036),and the differences were statistically significant( t = 3. 996,12. 230,23. 130; with P values below 0. 05). The protein expression levels of β-catenin,Wnt3 a,SDF-1 were higher in the porcine ADM wound group( 0. 557 ±0. 009,0. 626 ± 0. 066,0. 868 ± 0. 102) than in the gauze wound group( 0. 302 ± 0. 010,0. 109 ± 0. 019,0. 036 ± 0. 009),and the differences were statistically significant( t = 32. 830,13. 020,14. 130; with P values below 0. 05). And the protein expression levels of Wnt3 a,SDF-1 in the porcine ADM wound margin group( 0. 419 ± 0. 014,0. 370 ± 0. 069) were higher than those in the gauze margin group( 0. 115 ± 0. 020,0. 056 ± 0. 007),and the differences were statistically significant( t = 21. 460,7. 825; with P values below0. 05). Immunohistochemistry results showed that Wnt3 a,SDF-1,FGF2 and FGF9 in the porcine ADM group were higher than those in the gauze group,and the positive cells were mainly distributed around hair follicle cells. Conclusion In the wound repair process,porcine ADM may promotes the regeneration of hair follicles by up-regulating the expression of SDF-1 and Wnt3 a/β-catenin signaling pathway.
引文
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[16] Amini-Nik S,Glancy D,Boimer C,et al. Pax7 expressing cells contribute to dermal wound repair,regulating scar size through aβ-catenin mediated process[J]. Stem Cells,2011,29(9):1371-1379.
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[18] Fei Y,Xiao L,Doetschman T,et al. Fibroblast growth factor 2stimulation of osteoblast differentiation and bone formation is mediated by modulation of the Wnt signaling pathway[J]. J Biol Chem,2011,286(47):40575-40583.
[19] Hung IH,Yu K,Lavine KJ, et al. FGF9 regulates early hypertrophic chondrocyte differentiation and skeletal vascularization in the developing stylopod[J]. Dev Biol,2007,307(2):300-313.
[20] Giannouli CC,Kletsas D. TGF-beta regulates differentially the proliferation of fetal and adult human skin fibroblasts via the activation of PKA and the autocrine action of FGF-2[J]. Cell Signal,2006,18(9):1417-1429.
[21] Cheon SS,Wei Q,Gurung A,et al. Beta-catenin regulates wound size and mediates the effect of TGF-beta in cutaneous healing[J]. FASEB J,2006,20(6):692-701.
[22] Wang X,Chen H,Tian R,et al. Macrophages induce AKT/β-catenin-dependent Lgr5+stem cell activation and hair follicle regeneration through TNF[J]. Nat Commun,2017,8:1409.
[2] Bondioli E, Fini M, Veronesi F, et al. Development and evaluation of decellularized membrane from human dermis[J]. J Tissue Eng Regen Med,2014,8(4):325-336.
[3] Eming SA,Hubbell JA. Extracellular matrix in angiogenesis:dynamic structures with translational potential[J]. Exp Dermatol,2011,20(7):605-613.
[4] Yan W,Liu H,Deng X,et al. Acellular dermal matrix scaffolds coated with connective tissue growth factor accelerate diabetic wound healing by increasing fibronectin through PKC signalling pathway[J]. J Tissue Eng Regen Med,2018,12(3):e1461-e1473.
[5] Nie C,Yang D,Morris SF. Local delivery of adipose-derived stem cells via acellular dermal matrix as a scaffold:a new promising strategy to accelerate wound healing[J]. Med Hypotheses,2009,72(6):679-682.
[6] Sigalove S. Options in Acellular Dermal Matrix-Device Assembly[J]. Plast Reconstr Surg,2017,140(6S Prepectoral Breast Reconstruction):39S-42S.
[7] Hughes OB,Rakosi A,Macquhae F,et al. A Review of Cellular and Acellular Matrix Products:Indications, Techniques, and Outcomes[J]. Plast Reconstr Surg,2016,138(3 Suppl):138S-147S.
[8] Zhang Z,Lv L,Mamat M,et al. Xenogenic(porcine)acellular dermal matrix is useful for the wound healing of severely damaged extremities[J]. Exp Therapeutic Med,2014,7(3):621-624.
[9]王永飞,程勇,许喜生.毛囊干细胞在创面愈合中的应用进展[J].现代医药卫生,2015,31(4):548-551.
[10] Bermudez DM,Xu J,Herdrich BJ,et al. Inhibition of stromal cell-derived factor-1αfurther impairs diabetic wound healing[J]. J Vasc Surg,2011,53(3):774-784.
[11] Nusse R. Wnt signaling and stem cell control[J]. Cell Res,2008,18(5):523-527.
[12] Clevers H,Nusse R. Wnt/β-catenin signaling and disease[J].Cell,2012,149(6):1192-1205.
[13] Kishimoto J,Burgeson RE,Morgan1 BA. Wnt signaling maint ains t he hai r-inducing act ivi ty of t he dermal papilla[J].Genes Dev,2000,14(10):1181-1185.
[14] Carre AL,James AW,Mac Leod L,et al. Interaction of wingless protein(Wnt), transforming growth factor-beta1, and hyaluronan production in fetal and postnatal fibroblasts[J]. PlastReconstr Surg,2010,25(1):74-88.
[15] Ito M,Yang Z,Andl T,et al. Wnt-dependent de novo hair follele regeneration in adult mouse skin wound after wounding[J]. Nature,2007,447(7142):316-320.
[16] Amini-Nik S,Glancy D,Boimer C,et al. Pax7 expressing cells contribute to dermal wound repair,regulating scar size through aβ-catenin mediated process[J]. Stem Cells,2011,29(9):1371-1379.
[17] Kanda S,Miyata Y,Kanetake H. Fibroblast growth factor-2-mediated capillary morphogenesis of endothelial cells requires signals via Flt-1/vascular endothelial growth factor receptor-1:possible involvement of c-Akt[J]. J Biol Chem,2004,279(6):4007-4016.
[18] Fei Y,Xiao L,Doetschman T,et al. Fibroblast growth factor 2stimulation of osteoblast differentiation and bone formation is mediated by modulation of the Wnt signaling pathway[J]. J Biol Chem,2011,286(47):40575-40583.
[19] Hung IH,Yu K,Lavine KJ, et al. FGF9 regulates early hypertrophic chondrocyte differentiation and skeletal vascularization in the developing stylopod[J]. Dev Biol,2007,307(2):300-313.
[20] Giannouli CC,Kletsas D. TGF-beta regulates differentially the proliferation of fetal and adult human skin fibroblasts via the activation of PKA and the autocrine action of FGF-2[J]. Cell Signal,2006,18(9):1417-1429.
[21] Cheon SS,Wei Q,Gurung A,et al. Beta-catenin regulates wound size and mediates the effect of TGF-beta in cutaneous healing[J]. FASEB J,2006,20(6):692-701.
[22] Wang X,Chen H,Tian R,et al. Macrophages induce AKT/β-catenin-dependent Lgr5+stem cell activation and hair follicle regeneration through TNF[J]. Nat Commun,2017,8:1409.