不同来源间充质干细胞条件培养基对内源性衰老细胞作用的比较
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摘要
背景:随着经济的发展,人们对美的追求越来越高,抗衰老研究成为当今的研究热点。干细胞是具有自我更新能力和多向分化能力的细胞,间充质干细胞是其中的一种。研究表明间充质干细胞能通过旁分泌作用修复损伤衰老的细胞,但对于不同来源间充质干细胞条件培养基的抗皮肤衰老作用研究甚少。目的:比较人脂肪、羊水、胎盘、脐带来源间充质干细胞条件培养基对自然衰老皮肤成纤维细胞的抗衰老作用。方法:制备人脂肪、羊水、胎盘、脐带4种不同来源的间充质干细胞条件培养基,添加到自然衰老皮肤成纤维细胞中,采用CCK-8法检测细胞活力、β-半乳糖苷酶染色试剂盒检测细胞衰老率、DCFH-DA探针检测活性氧含量以及q PCR检测p16、p53、COL1、KLOTHO基因表达量。结果与结论:①4种间充质干细胞条件培养基均能提高皮肤成纤维细胞的活力,其中脂肪间充质干细胞条件培养基的效果最好,β-半乳糖苷酶染色率最低;②4种间充质干细胞条件培养基均能降低活性氧含量,脂肪和羊水来源间充质干细胞条件培养基组的活性氧含量最低;③p16和p53在胎盘和脐带间充质干细胞条件培养基组的表达均高于对照组,在羊水来源间充质干细胞条件培养基组的表达较低,脂肪间充质干细胞条件培养基组p16表达显著低于对照组,但p53的表达较高;④COL1基因表达除羊水间充质干细胞条件培养基组外,其他3组均显著高于对照组,KLOTHO基因表达除胎盘间充质干细胞条件培养基组外,其他3组均显著高于对照组,且脂肪间充质干细胞条件培养基组的表达最高;⑤结果显示,4种不同来源间充质干细胞条件培养基能在一定程度上延缓细胞衰老,但作用效果存在差异,其作用机制还有待进一步研究。
BACKGROUND: With the economic development, people have an increasing pursuit of beauty, and anti-aging research has become a hot research topic. Stem cells have self-renewal and multi-directional differentiation ability, and mesenchymal stem cells are one of them. Studies have shown that mesenchymal stem cells can repair aging cells through paracrine action, but little has been reported on the anti-aging effects of mesenchymal stem cell conditioned media from different sources.OBJECTIVE: To compare the anti-aging effects of human adipose-, amniotic fluid-, placenta-and umbilical cord-derived mesenchymal stem cell conditioned media on aging skin fibroblasts.METHODS: Human adipose, amniotic fluid, placenta and umbilical cord-derived mesenchymal stem cell conditioned media were prepared and used to culture aging skin fibroblasts. Cell viability was detected by cell counting kit-8 and cell senescence rate measured byβ-galactosidase staining kit. DCFH-DA probe was used to detect the content of reactive oxygen species. qPCR was used to detect the expression levels of p16, p53, COL1 and KLOTHO genes.RESULTS AND CONCLUSION:(1) Four sources of mesenchymal stem cell conditioned media could increase the cell viability of skin fibroblasts, among which the adipose-derived mesenchymal stem cell conditioned medium had the best effect and the lowest β-galactosidase staining rate.(2) Four sources of mesenchymal stem cell conditioned media could reduce reactive oxygen species content, and adipose-and amniotic fluid-derived mesenchymal stem cell conditioned media had the lowest content of reactive oxygen species.(3) p16 and p53 levels in the placenta and umbilical cord-derived mesenchymal stem cell conditioned media were higher than those in the control group. The expression of p53 in the amniotic fluid-derived mesenchymal stem cell conditioned medium was lower than that in the control group, but there was no difference in the p16 expression between the two groups. Compared with the control group, the expression of p16 was lower and the expression of p53 was higher in the adipose-derived mesenchymal stem cell conditioned medium.(4) Compared with the control group, the expression of COL1 gene was significantly higher in the different culture media except for the amniotic fluid-derived mesenchymal stem cell conditioned medium. Compared with the control group, the expression of KLOTHO was significantly higher in different culture media except for placenta-derived mesenchymal stem cell conditioned medium, and highest in the adipose-derived mesenchymal stem cell conditioned medium. To conclude, mesenchymal stem cell conditioned media from four different sources can delay cell senescence to a certain extent,though its mechanism remains to be further studied.
BACKGROUND: With the economic development, people have an increasing pursuit of beauty, and anti-aging research has become a hot research topic. Stem cells have self-renewal and multi-directional differentiation ability, and mesenchymal stem cells are one of them. Studies have shown that mesenchymal stem cells can repair aging cells through paracrine action, but little has been reported on the anti-aging effects of mesenchymal stem cell conditioned media from different sources.OBJECTIVE: To compare the anti-aging effects of human adipose-, amniotic fluid-, placenta-and umbilical cord-derived mesenchymal stem cell conditioned media on aging skin fibroblasts.METHODS: Human adipose, amniotic fluid, placenta and umbilical cord-derived mesenchymal stem cell conditioned media were prepared and used to culture aging skin fibroblasts. Cell viability was detected by cell counting kit-8 and cell senescence rate measured byβ-galactosidase staining kit. DCFH-DA probe was used to detect the content of reactive oxygen species. qPCR was used to detect the expression levels of p16, p53, COL1 and KLOTHO genes.RESULTS AND CONCLUSION:(1) Four sources of mesenchymal stem cell conditioned media could increase the cell viability of skin fibroblasts, among which the adipose-derived mesenchymal stem cell conditioned medium had the best effect and the lowest β-galactosidase staining rate.(2) Four sources of mesenchymal stem cell conditioned media could reduce reactive oxygen species content, and adipose-and amniotic fluid-derived mesenchymal stem cell conditioned media had the lowest content of reactive oxygen species.(3) p16 and p53 levels in the placenta and umbilical cord-derived mesenchymal stem cell conditioned media were higher than those in the control group. The expression of p53 in the amniotic fluid-derived mesenchymal stem cell conditioned medium was lower than that in the control group, but there was no difference in the p16 expression between the two groups. Compared with the control group, the expression of p16 was lower and the expression of p53 was higher in the adipose-derived mesenchymal stem cell conditioned medium.(4) Compared with the control group, the expression of COL1 gene was significantly higher in the different culture media except for the amniotic fluid-derived mesenchymal stem cell conditioned medium. Compared with the control group, the expression of KLOTHO was significantly higher in different culture media except for placenta-derived mesenchymal stem cell conditioned medium, and highest in the adipose-derived mesenchymal stem cell conditioned medium. To conclude, mesenchymal stem cell conditioned media from four different sources can delay cell senescence to a certain extent,though its mechanism remains to be further studied.
引文
[1]Fernández JR, Webb C, Rouzard K, et al. SIG-1273 protects skin against urban air pollution and when formulated in AgeIQ?Night Cream anti-aging benefits clinically demonstrated. J Cosmet Dermatol. 2018 Nov 19. doi:10.1111/jocd.12825.[Epub ahead of print]
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[3]Li H, Gao A, Jiang N, et al. Protective Effect of Curcumin Against Acute Ultraviolet B Irradiation-induced Photo-damage.Photochem Photobiol. 2016;92(6):808-815.
[4]Baek B, Lee SH, Kim K, et al. Ellagic acid plays a protective role against UV-B-induced oxidative stress by up-regulating antioxidant components in human dermal fibroblasts. Korean J Physiol Pharmacol. 2016;20(3):269-277.
[5]Uder C, Brückner S, Winkler S, et al. Mammalian MSC from selected species:Features and applications. Cytometry A.2018;93(1):32-49.
[6]詹小舒,罗冬章,王丙云,等.犬脐带间充质干细胞来源外泌体修复皮肤创伤[J].中国组织工程研究,2018,22(25):4021-4027.
[7]张贝莹,王丙云,罗冬章,等.犬脐带间充质干细胞抗关节炎炎症反应的研究[J].中国兽医杂志,2018,54(6):16-20.
[8]张贝莹,罗冬章,麦海涛,等.骨髓间充质干细胞移植修复皮肤创伤[J].中国组织工程研究,2017,21(29):4611-4616.
[9]Kilroy GE, Foster SJ, Wu X, et al. Cytokine profile of human adipose-derived stem cells:expression of angiogenic,hematopoietic, and pro-inflammatory factors. J Cell Physiol.2007;212(3):702-709.
[10]Ertl J, Pichlsberger M, Tuca AC, et al. Comparative study of regenerative effects of mesenchymal stem cells derived from placental amnion, chorion and umbilical cord on dermal wounds. Placenta. 2018;65:37-46.
[11]孙青.羊水干细胞在皮肤损伤修复中的作用及机制研究[D].苏州:苏州大学,2016.
[12]Lee SC, Jeong HJ, Lee SK, et al. Hypoxic Conditioned Medium From Human Adipose-Derived Stem Cells Promotes Mouse Liver Regeneration Through JAK/STAT3 Signaling.Stem Cells Transl Med. 2016;5(6):816-825.
[13]Ding C, Zou Q, Wang F, et al. HGF and BFGF Secretion by Human Adipose-Derived Stem Cells Improves Ovarian Function During Natural Aging via Activation of the SIRT1/FOXO1 Signaling Pathway. Cell Physiol Biochem.2018;45(4):1316-1332.
[14]Cooper DR, Wang C, Patel R, et al. Human Adipose-Derived Stem Cell Conditioned Media and Exosomes Containing MALAT1 Promote Human Dermal Fibroblast Migration and Ischemic Wound Healing. Adv Wound Care(New Rochelle).2018;7(9):299-308.
[15]Kwon TR, Oh CT, Choi EJ, et al. Conditioned medium from human bone marrow-derived mesenchymal stem cells promotes skin moisturization and effacement of wrinkles in UVB-irradiated SKH-1 hairless mice. Photodermatol Photoimmunol Photomed. 2016;32(3):120-128.
[16]Kamolz LP, Keck M, Kasper C. Wharton's jelly mesenchymal stem cells promote wound healing and tissue regeneration.Stem Cell Res Ther. 2014;5(3):62.
[17]赵姝灿,郑桂纯,林连蓬,等.人羊水、脐带、胎盘来源间充质干细胞体外增殖、分化、运输和免疫学特性的比较[J].中国组织工程研究,2018,22(25):4028-4034.
[18]Xu S, Cai Y, Wei Y. mTOR Signaling from Cellular Senescence to Organismal Aging. Aging Dis. 2013;5(4):263-273.
[19]彭晓敏. miR-377通过抑制DNMT1促进人皮肤成纤维细胞衰老[D].长沙:中南大学,2013.
[20]赵传祥,李宏慧,汤思熠,等.间充质干细胞培养上清液抑制成纤维细胞的衰老[J].江苏大学学报(医学版),2019,29(1):12-16.
[21]陈迪,章漳,蒋耀权,等.冰川水和白雪茶提取物延缓人真皮成纤维细胞衰老的实验研究[J].天然产物研究与开发, 2018,30(3):469-474.
[22]Xiong ZM, O'Donovan M, Sun L, et al. Anti-Aging Potentials of Methylene Blue for Human Skin Longevity. Sci Rep. 2017;7(1):2475.
[23]Kim EJ, Kim YK, Kim MK, et al. UV-induced inhibition of adipokine production in subcutaneous fat aggravates dermal matrix degradation in human skin. Sci Rep. 2016;6:25616.
[24]Di Lullo GA, Sweeney SM, Korkko J, et al. Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type I collagen. J Biol Chem. 2002;277(6):4223-4231.
[25]Park YM, Park SN. Inhibitory Effect of Lupeol on MMPs Expression using Aged Fibroblast through Repeated UVA Irradiation. Photochem Photobiol. 2019;95(2):587-594.
[26]Fulop GA, Kiss T, Tarantini S, et al. Nrf2 deficiency in aged mice exacerbates cellular senescence promoting cerebrovascular inflammation. Geroscience. 2018;40(5-6):513-521.
[27]常雅琦,文敏,赵华,等.Klotho蛋白对氧化应激的影响及可能机制[J].动物营养学报,2018,30(9):3459-3465.
[28]Kuro-O M. The Klotho proteins in health and disease. Nat Rev Nephrol. 2019;15(1):27-44.
[29]Cui W, Leng B, Wang G. Klotho protein inhibits H2O2-induced oxidative injury in endothelial Cells via regulation of PI3K/Akt/Nrf2/HO-1 Pathways. Can J Physiol Pharmacol.2018 Dec 21. doi:10.1139/cjpp-2018-0277.[Epub ahead of print]
[30]郭吉安.脂肪干细胞微囊泡抗皮肤成纤维细胞衰老作用的研究[D].镇江:江苏大学,2017.
[2]Tobin DJ. Introduction to skin aging. J Tissue Viability. 2017;26(1):37-46.
[3]Li H, Gao A, Jiang N, et al. Protective Effect of Curcumin Against Acute Ultraviolet B Irradiation-induced Photo-damage.Photochem Photobiol. 2016;92(6):808-815.
[4]Baek B, Lee SH, Kim K, et al. Ellagic acid plays a protective role against UV-B-induced oxidative stress by up-regulating antioxidant components in human dermal fibroblasts. Korean J Physiol Pharmacol. 2016;20(3):269-277.
[5]Uder C, Brückner S, Winkler S, et al. Mammalian MSC from selected species:Features and applications. Cytometry A.2018;93(1):32-49.
[6]詹小舒,罗冬章,王丙云,等.犬脐带间充质干细胞来源外泌体修复皮肤创伤[J].中国组织工程研究,2018,22(25):4021-4027.
[7]张贝莹,王丙云,罗冬章,等.犬脐带间充质干细胞抗关节炎炎症反应的研究[J].中国兽医杂志,2018,54(6):16-20.
[8]张贝莹,罗冬章,麦海涛,等.骨髓间充质干细胞移植修复皮肤创伤[J].中国组织工程研究,2017,21(29):4611-4616.
[9]Kilroy GE, Foster SJ, Wu X, et al. Cytokine profile of human adipose-derived stem cells:expression of angiogenic,hematopoietic, and pro-inflammatory factors. J Cell Physiol.2007;212(3):702-709.
[10]Ertl J, Pichlsberger M, Tuca AC, et al. Comparative study of regenerative effects of mesenchymal stem cells derived from placental amnion, chorion and umbilical cord on dermal wounds. Placenta. 2018;65:37-46.
[11]孙青.羊水干细胞在皮肤损伤修复中的作用及机制研究[D].苏州:苏州大学,2016.
[12]Lee SC, Jeong HJ, Lee SK, et al. Hypoxic Conditioned Medium From Human Adipose-Derived Stem Cells Promotes Mouse Liver Regeneration Through JAK/STAT3 Signaling.Stem Cells Transl Med. 2016;5(6):816-825.
[13]Ding C, Zou Q, Wang F, et al. HGF and BFGF Secretion by Human Adipose-Derived Stem Cells Improves Ovarian Function During Natural Aging via Activation of the SIRT1/FOXO1 Signaling Pathway. Cell Physiol Biochem.2018;45(4):1316-1332.
[14]Cooper DR, Wang C, Patel R, et al. Human Adipose-Derived Stem Cell Conditioned Media and Exosomes Containing MALAT1 Promote Human Dermal Fibroblast Migration and Ischemic Wound Healing. Adv Wound Care(New Rochelle).2018;7(9):299-308.
[15]Kwon TR, Oh CT, Choi EJ, et al. Conditioned medium from human bone marrow-derived mesenchymal stem cells promotes skin moisturization and effacement of wrinkles in UVB-irradiated SKH-1 hairless mice. Photodermatol Photoimmunol Photomed. 2016;32(3):120-128.
[16]Kamolz LP, Keck M, Kasper C. Wharton's jelly mesenchymal stem cells promote wound healing and tissue regeneration.Stem Cell Res Ther. 2014;5(3):62.
[17]赵姝灿,郑桂纯,林连蓬,等.人羊水、脐带、胎盘来源间充质干细胞体外增殖、分化、运输和免疫学特性的比较[J].中国组织工程研究,2018,22(25):4028-4034.
[18]Xu S, Cai Y, Wei Y. mTOR Signaling from Cellular Senescence to Organismal Aging. Aging Dis. 2013;5(4):263-273.
[19]彭晓敏. miR-377通过抑制DNMT1促进人皮肤成纤维细胞衰老[D].长沙:中南大学,2013.
[20]赵传祥,李宏慧,汤思熠,等.间充质干细胞培养上清液抑制成纤维细胞的衰老[J].江苏大学学报(医学版),2019,29(1):12-16.
[21]陈迪,章漳,蒋耀权,等.冰川水和白雪茶提取物延缓人真皮成纤维细胞衰老的实验研究[J].天然产物研究与开发, 2018,30(3):469-474.
[22]Xiong ZM, O'Donovan M, Sun L, et al. Anti-Aging Potentials of Methylene Blue for Human Skin Longevity. Sci Rep. 2017;7(1):2475.
[23]Kim EJ, Kim YK, Kim MK, et al. UV-induced inhibition of adipokine production in subcutaneous fat aggravates dermal matrix degradation in human skin. Sci Rep. 2016;6:25616.
[24]Di Lullo GA, Sweeney SM, Korkko J, et al. Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type I collagen. J Biol Chem. 2002;277(6):4223-4231.
[25]Park YM, Park SN. Inhibitory Effect of Lupeol on MMPs Expression using Aged Fibroblast through Repeated UVA Irradiation. Photochem Photobiol. 2019;95(2):587-594.
[26]Fulop GA, Kiss T, Tarantini S, et al. Nrf2 deficiency in aged mice exacerbates cellular senescence promoting cerebrovascular inflammation. Geroscience. 2018;40(5-6):513-521.
[27]常雅琦,文敏,赵华,等.Klotho蛋白对氧化应激的影响及可能机制[J].动物营养学报,2018,30(9):3459-3465.
[28]Kuro-O M. The Klotho proteins in health and disease. Nat Rev Nephrol. 2019;15(1):27-44.
[29]Cui W, Leng B, Wang G. Klotho protein inhibits H2O2-induced oxidative injury in endothelial Cells via regulation of PI3K/Akt/Nrf2/HO-1 Pathways. Can J Physiol Pharmacol.2018 Dec 21. doi:10.1139/cjpp-2018-0277.[Epub ahead of print]
[30]郭吉安.脂肪干细胞微囊泡抗皮肤成纤维细胞衰老作用的研究[D].镇江:江苏大学,2017.
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