人脐带间充质干细胞对人真皮成纤维细胞增殖和细胞外基质基因表达的影响
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摘要
目的探讨人脐带间充质干细胞(huc MSCs)对人真皮成纤维细胞(HDFib)增殖与细胞外基质相关基因表达的影响及其可能机制。方法采用II型胶原酶消化法从新生儿脐带中分离制备huc MSCs,用流式细胞术鉴定P_3代huc MSCs表面标记抗原,用油红O染色、茜素红S染色和Masson染色进行间充质干细胞三向分化能力鉴定;随后与P_3代HDFib进行transwell共培养,对照组transwell小室上添加等量培养基,观察24、48和72 h时两组HDFib的细胞形态,CCK8法进行细胞增殖检测,q RT-PCR法测定培养24和48h时HDFib主要细胞外基质蛋白collagenI、elastin、fibronectin、MMP-1和细胞信号转导相关基因PI3K、Akt、p38和caspase3 mRNA表达水平。结果制备获得的hucMSCs呈典型成纤维细胞样并贴壁生长,其表面间充质干细胞标志性抗原CD105、CD90、CD73阳性表达率分别为96.97%、99.75%和99.81%,诱导培养16d后出现明显的成脂、成骨和成软骨细胞特性。huc MSCs共培养处理24、48和72 h,HDFib细胞存活率较对照组分别显著增加了7.29%(P<0.01)、25.82%(P <0.05)和11.74%(P <0.05),与形态学观察结果一致。q RT-PCR结果显示,huc MSCs共培养处理24h,HDFib elastin和fibronectin基因表达分别上调为对照组的1.45倍(P <0.01)和1.30倍(P <0.05),PI3K mRNA水平上调为对照组的1.19倍(P <0.05);共培养48 h时,collagen I、elastin、fibronectin和PI3K、Akt分别上调2.60倍(P<0.001)、3.66倍(P <0.05)、3.45倍(P <0.01)和1.43倍(P <0.05)、1.65倍(P <0.05),MMP-1、p38和caspase3转录水平显著降低至对照组的0.70倍(P <0.01)、0.60倍(P <0.01)和0.86倍(P <0.05)。结论 huc MSCs能够显著促进正常HDFib增殖,同时提高HDFibI型胶原、弹性蛋白和纤维粘连蛋白转录水平,是理想的皮肤组织工程学种子细胞。hucMSCs对HDFib细胞外基质的调节作用可能与激活HDFib皮肤创伤修复信号通路PI3K/Akt、下调细胞外基质调节基因p38、凋亡基因caspase3的m RNA水平有关。
Objective To investigate the effects of human umbilical cord mesenchymal stem cells(hucMSCs) on the cell proliferation and extracellular matrix genes expression of normal human dermal fibroblast(HDFib) and potential mechanisms.Methods The hucMSCs were derived from healthy full-term newborn umbilical cord through a collagenase digestion method.Flow cytometry was applied to determine the characteristic mesenchymal stem cell antigenic markers of P_3-generation hucMSCs,and oil red O, alizarin red S and Masson staining were used for testing its adipogenic, osteogenic and chondrogenic differentiation capacities, respectively. Then, P_3-generation HDFib were treated with hucMSCs by transwell experiment(co-culture group), while equal volume of medium was added to the control group. After a period of 24, 48 and 72 h co-culture, the proliferation of HDFib was measured by CCK8 assay and cell morphology were also recorded. The mainly extracellular matrix proteins, collagen I, elastin,fibronectin, matrix metalloproteinase-1(MMP-1) and cellular signal transduction pathways PI3 K, Akt, p38 and caspase3 mRNA expression after 24 and 48 h culture were further characterized by qRT-PCR.Results The adherent hucMSCs showed the shape of fibroblast, which could differentiate into osteoblasts, adipocytes and chondrocytes after 16 d induction culture period in vitro. The expression of CD105, CD90 and CD73 on hucMSCs were 96.97%,99.75% and 99.81%, respectively. Consistent with the morphologic results, the viability of HDFib treated with hucMSCs was significantly increased for 7.29%(P < 0.01), 25.82%(P < 0.05) and 11.74%(P < 0.05) after 24, 48 and 72 h co-culture, respectively.Moreover, elastin, fibronectin and PI3 K gene expression of HDFib were up-regulated by 1.45(P < 0.01), 1.30(P < 0.05) and 1.19(P < 0.05) folds after 24 h co-culture with hucMSCs compared to the control group, and collagen I, elastin, fibronectin, PI3 K and Akt mRNA level were up-regulated by 2.60(P < 0.001), 3.66(P < 0.05), 3.45(P < 0.01), 1.43(P < 0.05) and 1.65(P < 0.05) folds after48 h co-culture, while MMP-1, p38 and caspase3 were down-regulated by 0.70(P < 0.01), 0.60(P < 0.01) and 0.86(P < 0.05) folds of control.Conclusion Our results show that hucMSCs can significantly promote the proliferation of HDFib and increase the transcription levels of collagen I, elastin and fibronectin, which indicates that hucMSCs are potential seed cells for skin tissue engineering. Besides,our results indicate that the regulation of hucMSCs on HDFib extracellular matrix may be related to the activation of skin wound repair pathway PI3 K/Akt and the decrease of p38 and caspase3 expression.
Objective To investigate the effects of human umbilical cord mesenchymal stem cells(hucMSCs) on the cell proliferation and extracellular matrix genes expression of normal human dermal fibroblast(HDFib) and potential mechanisms.Methods The hucMSCs were derived from healthy full-term newborn umbilical cord through a collagenase digestion method.Flow cytometry was applied to determine the characteristic mesenchymal stem cell antigenic markers of P_3-generation hucMSCs,and oil red O, alizarin red S and Masson staining were used for testing its adipogenic, osteogenic and chondrogenic differentiation capacities, respectively. Then, P_3-generation HDFib were treated with hucMSCs by transwell experiment(co-culture group), while equal volume of medium was added to the control group. After a period of 24, 48 and 72 h co-culture, the proliferation of HDFib was measured by CCK8 assay and cell morphology were also recorded. The mainly extracellular matrix proteins, collagen I, elastin,fibronectin, matrix metalloproteinase-1(MMP-1) and cellular signal transduction pathways PI3 K, Akt, p38 and caspase3 mRNA expression after 24 and 48 h culture were further characterized by qRT-PCR.Results The adherent hucMSCs showed the shape of fibroblast, which could differentiate into osteoblasts, adipocytes and chondrocytes after 16 d induction culture period in vitro. The expression of CD105, CD90 and CD73 on hucMSCs were 96.97%,99.75% and 99.81%, respectively. Consistent with the morphologic results, the viability of HDFib treated with hucMSCs was significantly increased for 7.29%(P < 0.01), 25.82%(P < 0.05) and 11.74%(P < 0.05) after 24, 48 and 72 h co-culture, respectively.Moreover, elastin, fibronectin and PI3 K gene expression of HDFib were up-regulated by 1.45(P < 0.01), 1.30(P < 0.05) and 1.19(P < 0.05) folds after 24 h co-culture with hucMSCs compared to the control group, and collagen I, elastin, fibronectin, PI3 K and Akt mRNA level were up-regulated by 2.60(P < 0.001), 3.66(P < 0.05), 3.45(P < 0.01), 1.43(P < 0.05) and 1.65(P < 0.05) folds after48 h co-culture, while MMP-1, p38 and caspase3 were down-regulated by 0.70(P < 0.01), 0.60(P < 0.01) and 0.86(P < 0.05) folds of control.Conclusion Our results show that hucMSCs can significantly promote the proliferation of HDFib and increase the transcription levels of collagen I, elastin and fibronectin, which indicates that hucMSCs are potential seed cells for skin tissue engineering. Besides,our results indicate that the regulation of hucMSCs on HDFib extracellular matrix may be related to the activation of skin wound repair pathway PI3 K/Akt and the decrease of p38 and caspase3 expression.
引文
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[2]Chang Y,Li H,Guo Z.Mesenchymal stem cell-like properties in fibroblasts.Cell Physiol Biochem,2014,34(3):703-714.
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[6]Liu M,Wang JS,Liu MY,et al.Study of immunomodulatory function of exosomes derived from human umbilical cord mesenchymal stem cells.Natl Med J China,2015,95(32):2630-2633.(in Chinese)刘明,汪劲松,刘沐芸,等.人脐带间充质干细胞外泌体免疫调节功能的研究.中华医学杂志,2015,95(32):2630-2633.
[7]Zhang Y,Chen KJ,Li P,et al.Effects of cryopreservation methods on human pMSCs:programmed versus stepwise freezing.Chin Med Biotechnol,2017,12(6):515-519.(in Chinese)章毅,陈侃俊,李萍,等.程控降温法与二步法对胎盘间充质干细胞冻存效果的影响.中国医药生物技术,2017,12(6):513-519.
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[9]Yannas IV,Tzeranis D,So PT.Surface biology of collagen scaffold explains blocking of wound contraction and regeneration of skin and peripheral nerves.Biomed Mater,2015,11(1):014106.
[10]Quan T,Little E,Quan H,et al.Elevated matrix metalloproteinases and collagen fragmentation in photodamaged human skin:impact of altered extracellular matrix microenvironment on dermal fibroblast function.J Invest Dermatol,2013,133(5):1362-1366.
[11]Dang QF,Liu H,Yan JQ,et al.Characterization of collagen from haddock skin and wound healing properties of its hydrolysates.Biomed Mater,2015,10(1):015022.
[12]Mohamed MA,Jung M,Lee SM,et al.Protective effect of disporum sessile D.Don extract against UVB-induced photoaging via suppressing MMP-1 expression and collagen degradation in human skin cells.J Photochem Photobiol B,2014,133:73-79.
[13]Agren MS,Schnabel R,Christensen LH,et al.Tumor necrosis factor-alpha-accelerated degradation of type I collagen in human skin is associated with elevated matrix metalloproteinase(MMP)-1 and MMP-3 ex vivo.Eur J Cell Biol,2015,94(1):12-21.
[14]Mora Huertas AC,Schmelzer CEH,Luise C,et al.Degradation of tropoelastin and skin elastin by neprilysin.Biochimie,2018,146:73-78.
[15]Jeon YK,Jang YH,Yoo DR,et al.Mesenchymal stem cells'interaction with skin:wound-healing effect on fibroblast cells and skin tissue.Wound Repair Regen,2010,18(6):655-661.
[16]Arno AI,Amini-Nik S,Blit PH,et al.Effect of human Wharton's jelly mesenchymal stem cell paracrine signaling on keloid fibroblasts.Stem Cells Transl Med,2014,3(3):299-307.
[17]Sato C,Yamamoto Y,Funayama E,et al.Conditioned medium obtained from amnion-derived mesenchymal stem cell culture prevents activation of keloid fibroblasts.Plast Reconstr Surg,2018,141(2):390-398.
[18]Li X,Hamada T,Ohata C,et al.Potential mesenchymal stem cell therapy for skin diseases.Exp Dermatol,2013,22(8):515-516.
[19]Shabbir A,Cox A,Rodriguez-Menocal L,et al.Mesenchymal stem cell exosomes induce proliferation and migration of normal and chronic wound fibroblasts,and enhance angiogenesis in vitro.Stem Cells Dev,2015,24(14):1635-1647.
[20]Sarojini H,Estrada R,Lu H,et al.PEDF from mouse mesenchymal stem cell secretome attracts fibroblasts.J Cell Biochem,2008,104(5):1793-1802.
[21]Kim YJ,Yoo SM,Park HH,et al.Exosomes derived from human umbilical cord blood mesenchymal stem cells stimulates rejuvenation of human skin.Biochem Biophys Res Commun,2017,493(2):1102-1108.
[22]Zhang JJ,Guan DW,Yang DL.A study on the time-dependent expression of phospho-p38MAPK during the skin incised wound healing in mice.J Forensic Med,2007,23(5):321-324,cover 4.(in Chinese)张建军,官大威,杨大利.小鼠皮肤切创愈合过程中磷酸化p38MAPK表达及其与时间的相关性.法医学杂志,2007,23(5):321-324,封4.
[23]Pan M.Photoprotection effects of EGCG on reducing protein secretion and mRNA expression of MMP-1 in human skin fibroblasts after UVB or UVA irradiation and the relevant molecules mechanisms.Nanjing:Nanjing Medical University,2008.(in Chinese)潘敏.EGCG抑制UVB及UVA诱导的人皮肤成纤维细胞MMP-1水平及其相关分子机制的实验研究.南京:南京医科大学,2008.
[2]Chang Y,Li H,Guo Z.Mesenchymal stem cell-like properties in fibroblasts.Cell Physiol Biochem,2014,34(3):703-714.
[3]Zeng TF,Xin GH,He Y.Research progress of bone marrow mesenchymal stem cells on skin injury repairing.Chin J Clin(Electronic Ed),2011,5(16):4831-4834.(in Chinese)曾逃方,辛国华,何勇.应用骨髓间充质干细胞修复皮肤损伤的研究进展.中华临床医师杂志(电子版),2011,5(16):4831-4834.
[4]Trial J,Entman ML,Cieslik KA.Mesenchymal stem cell-derived inflammatory fibroblasts mediate interstitial fibrosis in the aging heart.J Mol Cell Cardiol,2016,91:28-34.
[5]Linard C,Tissedre F,Busson E,et al.Therapeutic potential of gingival fibroblasts for cutaneous radiation syndrome:comparison to bone marrow-mesenchymal stem cell grafts.Stem Cells Dev,2015,24(10):1182-1193.
[6]Liu M,Wang JS,Liu MY,et al.Study of immunomodulatory function of exosomes derived from human umbilical cord mesenchymal stem cells.Natl Med J China,2015,95(32):2630-2633.(in Chinese)刘明,汪劲松,刘沐芸,等.人脐带间充质干细胞外泌体免疫调节功能的研究.中华医学杂志,2015,95(32):2630-2633.
[7]Zhang Y,Chen KJ,Li P,et al.Effects of cryopreservation methods on human pMSCs:programmed versus stepwise freezing.Chin Med Biotechnol,2017,12(6):515-519.(in Chinese)章毅,陈侃俊,李萍,等.程控降温法与二步法对胎盘间充质干细胞冻存效果的影响.中国医药生物技术,2017,12(6):513-519.
[8]Dominici M,Le Blanc K,Mueller I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement.Cytotherapy,2006,8(4):315-317.
[9]Yannas IV,Tzeranis D,So PT.Surface biology of collagen scaffold explains blocking of wound contraction and regeneration of skin and peripheral nerves.Biomed Mater,2015,11(1):014106.
[10]Quan T,Little E,Quan H,et al.Elevated matrix metalloproteinases and collagen fragmentation in photodamaged human skin:impact of altered extracellular matrix microenvironment on dermal fibroblast function.J Invest Dermatol,2013,133(5):1362-1366.
[11]Dang QF,Liu H,Yan JQ,et al.Characterization of collagen from haddock skin and wound healing properties of its hydrolysates.Biomed Mater,2015,10(1):015022.
[12]Mohamed MA,Jung M,Lee SM,et al.Protective effect of disporum sessile D.Don extract against UVB-induced photoaging via suppressing MMP-1 expression and collagen degradation in human skin cells.J Photochem Photobiol B,2014,133:73-79.
[13]Agren MS,Schnabel R,Christensen LH,et al.Tumor necrosis factor-alpha-accelerated degradation of type I collagen in human skin is associated with elevated matrix metalloproteinase(MMP)-1 and MMP-3 ex vivo.Eur J Cell Biol,2015,94(1):12-21.
[14]Mora Huertas AC,Schmelzer CEH,Luise C,et al.Degradation of tropoelastin and skin elastin by neprilysin.Biochimie,2018,146:73-78.
[15]Jeon YK,Jang YH,Yoo DR,et al.Mesenchymal stem cells'interaction with skin:wound-healing effect on fibroblast cells and skin tissue.Wound Repair Regen,2010,18(6):655-661.
[16]Arno AI,Amini-Nik S,Blit PH,et al.Effect of human Wharton's jelly mesenchymal stem cell paracrine signaling on keloid fibroblasts.Stem Cells Transl Med,2014,3(3):299-307.
[17]Sato C,Yamamoto Y,Funayama E,et al.Conditioned medium obtained from amnion-derived mesenchymal stem cell culture prevents activation of keloid fibroblasts.Plast Reconstr Surg,2018,141(2):390-398.
[18]Li X,Hamada T,Ohata C,et al.Potential mesenchymal stem cell therapy for skin diseases.Exp Dermatol,2013,22(8):515-516.
[19]Shabbir A,Cox A,Rodriguez-Menocal L,et al.Mesenchymal stem cell exosomes induce proliferation and migration of normal and chronic wound fibroblasts,and enhance angiogenesis in vitro.Stem Cells Dev,2015,24(14):1635-1647.
[20]Sarojini H,Estrada R,Lu H,et al.PEDF from mouse mesenchymal stem cell secretome attracts fibroblasts.J Cell Biochem,2008,104(5):1793-1802.
[21]Kim YJ,Yoo SM,Park HH,et al.Exosomes derived from human umbilical cord blood mesenchymal stem cells stimulates rejuvenation of human skin.Biochem Biophys Res Commun,2017,493(2):1102-1108.
[22]Zhang JJ,Guan DW,Yang DL.A study on the time-dependent expression of phospho-p38MAPK during the skin incised wound healing in mice.J Forensic Med,2007,23(5):321-324,cover 4.(in Chinese)张建军,官大威,杨大利.小鼠皮肤切创愈合过程中磷酸化p38MAPK表达及其与时间的相关性.法医学杂志,2007,23(5):321-324,封4.
[23]Pan M.Photoprotection effects of EGCG on reducing protein secretion and mRNA expression of MMP-1 in human skin fibroblasts after UVB or UVA irradiation and the relevant molecules mechanisms.Nanjing:Nanjing Medical University,2008.(in Chinese)潘敏.EGCG抑制UVB及UVA诱导的人皮肤成纤维细胞MMP-1水平及其相关分子机制的实验研究.南京:南京医科大学,2008.
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