脂肪来源干细胞对瘢痕疙瘩成纤维细胞增殖影响的分子机制
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摘要
背景:脂肪来源干细胞已广泛用于组织填充修复,但是其对皮肤瘢痕抑制和修复的作用机制还不清楚。目的:探讨脂肪来源干细胞对瘢痕疙瘩成纤维细胞生物活性的影响及分子机制。方法:取第3代人脂肪来源干细胞,分别以0,3×10~4,6×10~4,1.2×10~5/孔接种于Transwell小室的上室(0为对照组,只含细胞培养液),取第4代对数生长期的瘢痕疙瘩成纤维细胞,以6×104/孔接种于下室(脂肪干细胞与瘢痕疙瘩成纤维细胞比例分别为0.5∶1,1∶1和2∶1),进行人脂肪来源干细胞与瘢痕疙瘩成纤维细胞共培养,培养24 h取下室瘢痕疙瘩成纤维细胞进行相关指标检测。结果与结论:(1)人脂肪来源干细胞能够显著抑制瘢痕疙瘩成纤维细胞的增殖、迁移和胶原合成能力,并且随着脂肪干细胞所占比例的升高,抑制作用显著增强;(2)人脂肪来源干细胞能够显著促进瘢痕疙瘩成纤维细胞凋亡,并且随着脂肪干细胞所占比例的升高,促进作用显著增强;(3)Westernblot检测显示人脂肪来源干细胞能够显著抑制瘢痕疙瘩成纤维细胞p-ERK、Bcl-2和β-catenin蛋白表达;(4)结果表明,脂肪来源干细胞通过抑制ERK/β-catenin通路抑制瘢痕疙瘩成纤维细胞增殖、迁移,并促进凋亡。关键词:
BACKGROUND: Adipose-derived stem cells have been widely used for tissue filling and repair, but the mechanism underlying skin scar inhibition and repair is still unclear. OBJECTIVE: To explore the effect of adipose-derived stem cells on biological activity of keloid fibroblasts and its molecular mechanism. METHODS: Passage 3 human adipose-derived stem cells were seeded into the upper Transwell chamber at the density of 0, 3×10~4, 6×10~4, 1.2×10~5 per well(0 indicates control group). Passage 4 keloid fibroblasts at logarithmic growth phase were inoculated into the lower chamber at the density of 6×10~4 per well, and co-cultured with human adipose-derived stem cells at the ratio of 1:0.5, 1:1 and 1:2, respectively. After 24-hour co-culture, keloid fibroblasts from the lower chamber were used for index measurement. RESULTS AND CONCLUSION: Human adipose-derived stem cells markedly inhibited the proliferation, migration and collagen synthesis of keloid fibroblasts, and the inhibitory effect was significantly enhanced as the proportion of adipose-derived stem cells increased. Human adipose-derived stem cells also markedly promoted cell apoptosis in keloid fibroblasts, and this effect was enhanced as the proportion of adipose-derived stem cells increased. Western blot results showed that human adipose-derived stem cells significantly suppressed keloid fibroblasts p-ERK, Bcl2 and β-catenin protein expression. Overall, adipose-derived stem cells can inhibit the proliferation and migration but promote apoptosis of keloid fibroblasts by inhibiting the ERK/β-catenin pathway.
BACKGROUND: Adipose-derived stem cells have been widely used for tissue filling and repair, but the mechanism underlying skin scar inhibition and repair is still unclear. OBJECTIVE: To explore the effect of adipose-derived stem cells on biological activity of keloid fibroblasts and its molecular mechanism. METHODS: Passage 3 human adipose-derived stem cells were seeded into the upper Transwell chamber at the density of 0, 3×10~4, 6×10~4, 1.2×10~5 per well(0 indicates control group). Passage 4 keloid fibroblasts at logarithmic growth phase were inoculated into the lower chamber at the density of 6×10~4 per well, and co-cultured with human adipose-derived stem cells at the ratio of 1:0.5, 1:1 and 1:2, respectively. After 24-hour co-culture, keloid fibroblasts from the lower chamber were used for index measurement. RESULTS AND CONCLUSION: Human adipose-derived stem cells markedly inhibited the proliferation, migration and collagen synthesis of keloid fibroblasts, and the inhibitory effect was significantly enhanced as the proportion of adipose-derived stem cells increased. Human adipose-derived stem cells also markedly promoted cell apoptosis in keloid fibroblasts, and this effect was enhanced as the proportion of adipose-derived stem cells increased. Western blot results showed that human adipose-derived stem cells significantly suppressed keloid fibroblasts p-ERK, Bcl2 and β-catenin protein expression. Overall, adipose-derived stem cells can inhibit the proliferation and migration but promote apoptosis of keloid fibroblasts by inhibiting the ERK/β-catenin pathway.
引文
[1]Rog-Zielinska EA, Norris RA, Kohl P, et al. The Living Scar--Cardiac Fibroblasts and the Injured Heart. Trends Mol Med. 2016;22(2):99-114.
[2]Li Y, Shi S, Gao J, et al. Cryptotanshinone downregulates the profibrotic activities of hypertrophic scar fibroblasts and accelerates wound healing:A potential therapy for the reduction of skin scarring. Biomed Pharmacother. 2016;80:80-86.
[3]Harris WM, Zhang P, Plastini M, et al. Evaluation of function and recovery of adipose-derived stem cells after exposure to paclitaxel. Cytotherapy. 2017;19(2):211-221.
[4]Huang CZ, Yang XN, Liu DC, et al. Calcitonin Gene-Related Peptide-Induced Calcium Alginate Gel Combined with Adipose-Derived Stem Cells Differentiating to Osteoblasts.Cell Biochem Biophys. 2015;73(3):609-617.
[5]Goh BS, Che Omar SN, Ubaidah MA, et al. Chondrogenesis of human adipose derived stem cells for future microtia repair using co-culture technique. Acta Otolaryngol. 2017;137(4):432-441.
[6]Zhong J, Guo B, Xie J, et al. Crosstalk between adipose-derived stem cells and chondrocytes:when growth factors matter. Bone Res. 2016;4:15036.
[7]Wang WZ, Fang XH, Williams SJ, et al. The impact of short-term refrigeration of human lipoaspirate on adipose-derived stem cells and adipocytes. J Plast Reconstr Aesthet Surg. 2015;68(1):137-139.
[8]Ferng AS, Marsh KM, Fleming JM, et al. Adipose-derived human stem/stromal cells:comparative organ specific mitochondrial bioenergy profiles. Springerplus. 2016;5(1):2057.
[9]Kumai Y, Kobler JB, Park H, et al. Modulation of vocal fold scar fibroblasts by adipose-derived stem/stromal cells.Laryngoscope. 2010;120(2):330-337.
[10]Zhang J, Liu Z, Cao W, et al. Amentoflavone inhibits angiogenesis of endothelial cells and stimulates apoptosis in hypertrophic scar fibroblasts. Burns. 2014;40(5):922-929.
[11]He L, Marneros AG. Macrophages are essential for the early wound healing response and the formation of a fibrovascular scar. Am J Pathol. 2013;182(6):2407-2417.
[12]Bernabei P, Rigamonti L, Ariotti S, et al. Functional analysis of T lymphocytes infiltrating the dermis and epidermis of post-burn hypertrophic scar tissues. Burns. 1999;25(1):43-48.
[13]Gong YF, Zhang XM, Liu F, et al. Inhibitory effect of recombinant human endostatin on the proliferation of hypertrophic scar fibroblasts in a rabbit ear model. Eur J Pharmacol. 2016;791:647-654.
[14]Wang H, Chen Z, Li X, et al. TSG-6 treatment promoted apoptosis in human fibroblasts of pathological scar. Cell Mol Biol(Noisy-le-grand). 2016;62(6):33-37.
[15]Li ZJ, Kim SM. The application of the starfish hatching enzyme for the improvement of scar and keloid based on the fibroblast-populated collagen lattice. Appl Biochem Biotechnol.2014;173(4):989-1002.
[16]Liu D, Li G, Liu D, et al. Effects of tetrandrine on the synthesis of collagen and scar-derived fibroblast DNA. Zhonghua Shao Shang Za Zhi. 2001;17(4):222-224.
[17]Tsagias N, Kouzi-Koliakos K, Koliakos I, et al. Addition of adipose-derived stem cells in cord blood cultures stimulates their pluripotent differentiation. Transplant Proc. 2009;41(10):4340-4344.
[18]Pu CM, Liu CW, Liang CJ, et al. Adipose-Derived Stem Cells Protect Skin Flaps against Ischemia/Reperfusion Injury via IL-6 Expression. J Invest Dermatol. 2017;137(6):1353-1362.
[19]Payne NL, Sun G, McDonald C, et al. Human adipose-derived mesenchymal stem cells engineered to secrete IL-10 inhibit APC function and limit CNS autoimmunity. Brain Behav Immun. 2013;30:103-114.
[20]Razmkhah M, Jaberipour M, Erfani N, et al. Adipose derived stem cells(ASCs)isolated from breast cancer tissue express IL-4, IL-10 and TGF-β1 and upregulate expression of regulatory molecules on T cells:do they protect breast cancer cells from the immune response. Cell Immunol. 2011;266(2):116-122.
[21]Razmkhah M, Jaberipour M, Hosseini A, et al. Expression profile of IL-8 and growth factors in breast cancer cells and adipose-derived stem cells(ASCs)isolated from breast carcinoma. Cell Immunol. 2010;265(1):80-85.
[22]Cao JQ, Liang YY, Li YQ, et al. Adipose-derived stem cells enhance myogenic differentiation in the mdx mouse model of muscular dystrophy via paracrine signaling. Neural Regen Res. 2016;11(10):1638-1643.
[23]Harn HJ, Lin SZ, Hung SH, et al. Adipose-derived stem cells can abrogate chemical-induced liver fibrosis and facilitate recovery of liver function. Cell Transplant. 2012;21(12):2753-2764.
[24]Rivera-Valdés JJ, García-Ba?uelos J, Salazar-Montes A, et al.Human adipose derived stem cells regress fibrosis in a chronic renal fibrotic model induced by adenine. PLoS One.2017;12(12):e0187907.
[25]Zhao J, Shu B, Chen L, et al. Prostaglandin E2 inhibits collagen synthesis in dermal fibroblasts and prevents hypertrophic scar formation in vivo. Exp Dermatol. 2016;25(8):604-610.
[26]Tejiram S, Zhang J, Travis TE, et al. Compression therapy affects collagen type balance in hypertrophic scar. J Surg Res.2016;201(2):299-305.
[27]武艳,张春雷,刘阳,等.骨髓间充质干细胞条件培养液对瘢痕成纤维细胞生物活性的影响[J].中国组织工程研究, 2014,18(7):1009-1014.
[28]Chun Q, ZhiYong W, Fei S, et al. Dynamic biological changes in fibroblasts during hypertrophic scar formation and regression. Int Wound J. 2016;13(2):257-262.
[29]张鹏,纪亮,张翠香,等.基质金属蛋白酶促进瘢痕疙瘩成纤维细胞迁移及其意义[J].中国现代医学杂志,2013,23(3):11-14.
[30]余州,宋雅娟,苏映军,等.KU-0063794抑制增生性瘢痕成纤维细胞增殖及迁移的研究[J].中国美容整形外科杂志,2017,28(6):328-331.
[31]Lin TY, Fan CW, Maa MC, et al. Lipopolysaccharide-promoted proliferation of Caco-2 cells is mediated by c-Src induction and ERK activation. Biomedicine(Taipei). 2015;5(1):5.
[32]Sapkota M, Li L, Choi H, et al. Bromo-honaucin A inhibits osteoclastogenic differentiation in RAW 264. 7 cells via Akt and ERK signaling pathways. Eur J Pharmacol. 2015;769:100-109.
[33]Zhang Z, Liu P, Wang J, et al. Ubiquitin-conjugating enzyme E2C regulates apoptosis-dependent tumor progression of non-small cell lung cancer via ERK pathway. Med Oncol.2015;32(5):149.
[34]Zuo H, Lin T, Wang D, et al. RKIP Regulates Neural Cell Apoptosis Induced by Exposure to Microwave Radiation Partly Through the MEK/ERK/CREB Pathway. Mol Neurobiol.2015;51(3):1520-1529.
[35]沈利,余扬,马少林.A型肉毒毒素对增生性瘢痕成纤维细胞增殖、凋亡及对ERK/MAPK信号通路的影响[J].医学研究杂志, 2017,46(7):26-29.
[36]刘宏伟,程飚,付小兵,等.血管紧张素Ⅱ对增生性瘢痕成纤维细胞细胞外信号调节激酶活性的影响[J].中国美容医学杂志, 2007,16(7):874-877.
[37]吴江群,聂兴举,秦泽莲.氧化苦参碱对病理性瘢痕成纤维细胞增殖凋亡的影响[J].中国微创外科杂志,2011,11(3):259-263.
[38]唐世杰,胡素銮,王玉银.Bcl-2对病理性瘢痕成纤维细胞增殖活性及细胞凋亡的影响[J].汕头大学医学院学报, 2002,15(4):211-212.
[39]李响,刘宏伟,梁杰,等.异泽兰黄素抑制瘢痕组织成纤维细胞增殖的机制[J].中国老年学杂,2017,37(22):5495-5498.
[2]Li Y, Shi S, Gao J, et al. Cryptotanshinone downregulates the profibrotic activities of hypertrophic scar fibroblasts and accelerates wound healing:A potential therapy for the reduction of skin scarring. Biomed Pharmacother. 2016;80:80-86.
[3]Harris WM, Zhang P, Plastini M, et al. Evaluation of function and recovery of adipose-derived stem cells after exposure to paclitaxel. Cytotherapy. 2017;19(2):211-221.
[4]Huang CZ, Yang XN, Liu DC, et al. Calcitonin Gene-Related Peptide-Induced Calcium Alginate Gel Combined with Adipose-Derived Stem Cells Differentiating to Osteoblasts.Cell Biochem Biophys. 2015;73(3):609-617.
[5]Goh BS, Che Omar SN, Ubaidah MA, et al. Chondrogenesis of human adipose derived stem cells for future microtia repair using co-culture technique. Acta Otolaryngol. 2017;137(4):432-441.
[6]Zhong J, Guo B, Xie J, et al. Crosstalk between adipose-derived stem cells and chondrocytes:when growth factors matter. Bone Res. 2016;4:15036.
[7]Wang WZ, Fang XH, Williams SJ, et al. The impact of short-term refrigeration of human lipoaspirate on adipose-derived stem cells and adipocytes. J Plast Reconstr Aesthet Surg. 2015;68(1):137-139.
[8]Ferng AS, Marsh KM, Fleming JM, et al. Adipose-derived human stem/stromal cells:comparative organ specific mitochondrial bioenergy profiles. Springerplus. 2016;5(1):2057.
[9]Kumai Y, Kobler JB, Park H, et al. Modulation of vocal fold scar fibroblasts by adipose-derived stem/stromal cells.Laryngoscope. 2010;120(2):330-337.
[10]Zhang J, Liu Z, Cao W, et al. Amentoflavone inhibits angiogenesis of endothelial cells and stimulates apoptosis in hypertrophic scar fibroblasts. Burns. 2014;40(5):922-929.
[11]He L, Marneros AG. Macrophages are essential for the early wound healing response and the formation of a fibrovascular scar. Am J Pathol. 2013;182(6):2407-2417.
[12]Bernabei P, Rigamonti L, Ariotti S, et al. Functional analysis of T lymphocytes infiltrating the dermis and epidermis of post-burn hypertrophic scar tissues. Burns. 1999;25(1):43-48.
[13]Gong YF, Zhang XM, Liu F, et al. Inhibitory effect of recombinant human endostatin on the proliferation of hypertrophic scar fibroblasts in a rabbit ear model. Eur J Pharmacol. 2016;791:647-654.
[14]Wang H, Chen Z, Li X, et al. TSG-6 treatment promoted apoptosis in human fibroblasts of pathological scar. Cell Mol Biol(Noisy-le-grand). 2016;62(6):33-37.
[15]Li ZJ, Kim SM. The application of the starfish hatching enzyme for the improvement of scar and keloid based on the fibroblast-populated collagen lattice. Appl Biochem Biotechnol.2014;173(4):989-1002.
[16]Liu D, Li G, Liu D, et al. Effects of tetrandrine on the synthesis of collagen and scar-derived fibroblast DNA. Zhonghua Shao Shang Za Zhi. 2001;17(4):222-224.
[17]Tsagias N, Kouzi-Koliakos K, Koliakos I, et al. Addition of adipose-derived stem cells in cord blood cultures stimulates their pluripotent differentiation. Transplant Proc. 2009;41(10):4340-4344.
[18]Pu CM, Liu CW, Liang CJ, et al. Adipose-Derived Stem Cells Protect Skin Flaps against Ischemia/Reperfusion Injury via IL-6 Expression. J Invest Dermatol. 2017;137(6):1353-1362.
[19]Payne NL, Sun G, McDonald C, et al. Human adipose-derived mesenchymal stem cells engineered to secrete IL-10 inhibit APC function and limit CNS autoimmunity. Brain Behav Immun. 2013;30:103-114.
[20]Razmkhah M, Jaberipour M, Erfani N, et al. Adipose derived stem cells(ASCs)isolated from breast cancer tissue express IL-4, IL-10 and TGF-β1 and upregulate expression of regulatory molecules on T cells:do they protect breast cancer cells from the immune response. Cell Immunol. 2011;266(2):116-122.
[21]Razmkhah M, Jaberipour M, Hosseini A, et al. Expression profile of IL-8 and growth factors in breast cancer cells and adipose-derived stem cells(ASCs)isolated from breast carcinoma. Cell Immunol. 2010;265(1):80-85.
[22]Cao JQ, Liang YY, Li YQ, et al. Adipose-derived stem cells enhance myogenic differentiation in the mdx mouse model of muscular dystrophy via paracrine signaling. Neural Regen Res. 2016;11(10):1638-1643.
[23]Harn HJ, Lin SZ, Hung SH, et al. Adipose-derived stem cells can abrogate chemical-induced liver fibrosis and facilitate recovery of liver function. Cell Transplant. 2012;21(12):2753-2764.
[24]Rivera-Valdés JJ, García-Ba?uelos J, Salazar-Montes A, et al.Human adipose derived stem cells regress fibrosis in a chronic renal fibrotic model induced by adenine. PLoS One.2017;12(12):e0187907.
[25]Zhao J, Shu B, Chen L, et al. Prostaglandin E2 inhibits collagen synthesis in dermal fibroblasts and prevents hypertrophic scar formation in vivo. Exp Dermatol. 2016;25(8):604-610.
[26]Tejiram S, Zhang J, Travis TE, et al. Compression therapy affects collagen type balance in hypertrophic scar. J Surg Res.2016;201(2):299-305.
[27]武艳,张春雷,刘阳,等.骨髓间充质干细胞条件培养液对瘢痕成纤维细胞生物活性的影响[J].中国组织工程研究, 2014,18(7):1009-1014.
[28]Chun Q, ZhiYong W, Fei S, et al. Dynamic biological changes in fibroblasts during hypertrophic scar formation and regression. Int Wound J. 2016;13(2):257-262.
[29]张鹏,纪亮,张翠香,等.基质金属蛋白酶促进瘢痕疙瘩成纤维细胞迁移及其意义[J].中国现代医学杂志,2013,23(3):11-14.
[30]余州,宋雅娟,苏映军,等.KU-0063794抑制增生性瘢痕成纤维细胞增殖及迁移的研究[J].中国美容整形外科杂志,2017,28(6):328-331.
[31]Lin TY, Fan CW, Maa MC, et al. Lipopolysaccharide-promoted proliferation of Caco-2 cells is mediated by c-Src induction and ERK activation. Biomedicine(Taipei). 2015;5(1):5.
[32]Sapkota M, Li L, Choi H, et al. Bromo-honaucin A inhibits osteoclastogenic differentiation in RAW 264. 7 cells via Akt and ERK signaling pathways. Eur J Pharmacol. 2015;769:100-109.
[33]Zhang Z, Liu P, Wang J, et al. Ubiquitin-conjugating enzyme E2C regulates apoptosis-dependent tumor progression of non-small cell lung cancer via ERK pathway. Med Oncol.2015;32(5):149.
[34]Zuo H, Lin T, Wang D, et al. RKIP Regulates Neural Cell Apoptosis Induced by Exposure to Microwave Radiation Partly Through the MEK/ERK/CREB Pathway. Mol Neurobiol.2015;51(3):1520-1529.
[35]沈利,余扬,马少林.A型肉毒毒素对增生性瘢痕成纤维细胞增殖、凋亡及对ERK/MAPK信号通路的影响[J].医学研究杂志, 2017,46(7):26-29.
[36]刘宏伟,程飚,付小兵,等.血管紧张素Ⅱ对增生性瘢痕成纤维细胞细胞外信号调节激酶活性的影响[J].中国美容医学杂志, 2007,16(7):874-877.
[37]吴江群,聂兴举,秦泽莲.氧化苦参碱对病理性瘢痕成纤维细胞增殖凋亡的影响[J].中国微创外科杂志,2011,11(3):259-263.
[38]唐世杰,胡素銮,王玉银.Bcl-2对病理性瘢痕成纤维细胞增殖活性及细胞凋亡的影响[J].汕头大学医学院学报, 2002,15(4):211-212.
[39]李响,刘宏伟,梁杰,等.异泽兰黄素抑制瘢痕组织成纤维细胞增殖的机制[J].中国老年学杂,2017,37(22):5495-5498.