脂肪间充质干细胞条件培养液联合富血小板纤维蛋白修复小鼠皮肤损伤
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摘要
背景:富血小板纤维蛋白可促进软组织愈合,间充质干细胞分泌的可溶性因子具有促进皮肤再生的作用,两者联合应用促进皮肤损伤修复尚未见报道。目的:探讨脂肪间充质干细胞条件培养液联合富血小板纤维蛋白对小鼠皮肤创伤愈合的影响。方法:制作C57/BL6小鼠(西南医科大学实验动物中心提供)全层皮肤损伤模型,随机分为4组,每组10只。自造模后即开始治疗,各组分别涂抹脂肪间充质干细胞专用培养基、脂肪间充质干细胞条件培养液、富血小板纤维蛋白、脂肪间充质干细胞条件培养液+富血小板纤维蛋白,3次/d,连续7d。在治疗后的1,3,7,10 d测量创面大小并计算治疗后3,7 d的创面愈合率;治疗后第14天进行创面愈合的组织学分析。结果与结论:(1)与脂肪间充质干细胞专用培养基组相比,其他3组创面愈合时间缩短;(2)治疗后3,7 d,脂肪间充质干细胞条件培养液+富血小板纤维蛋白组的创面愈合率高于其他3组,脂肪间充质干细胞条件培养液组和富血小板纤维蛋白组的创面愈合率高于脂肪间充质干细胞专用培养基组,差异有显著性意义(P <0.05);(3)组织学分析显示,脂肪间充质干细胞条件培养液+富血小板纤维蛋白组小鼠创面白细胞及成纤维细胞聚集、血管新生、肉芽组织重塑、上皮化均比其他3组明显,伤口愈合效果最好;(4)结果表明,脂肪间充质干细胞条件培养液与富血小板纤维蛋白联合应用能有效促进小鼠皮肤损伤的修复,效果优于单独应用脂肪间充质干细胞条件培养液或富血小板纤维蛋白。
BACKGROUND: Platelet-rich fibrin can promote soft tissue healing, and soluble factors secreted by mesenchymal stem cells can promote skin regeneration, but their combination effects on skin repair are little reported. OBJECTIVE: To investigate the effect of conditioned medium of adipose tissue-derived mesenchymal stem cells combined with platelet-rich fibrin on the healing of skin wound in mice. METHODS: Full-thickness skin injury models were established in C57/BL6 mice and randomly divided into four groups, 10 mice in each group. After the modeling, each group was smeared with the specific medium of adipose tissue-derived mesenchymal stem cells, the conditioned medium of adipose tissue-derived mesenchymal stem cells, the platelet-rich fibrin, the conditioned medium of adipose tissue-derived mesenchymal stem cells combined with platelet-rich fibrin, thrice daily for 7 consecutive days. The wound size was measured at 1, 3, 7 and 10 days after treatment and the wound healing rate was calculated at 3 and 7 days after treatment. Histological analysis of wound healing was performed at 14 days after treatment. RESULTS AND CONCLUSION: Compared with the specific culture medium of adipose tissue-derived mesenchymal stem cells, the wound healing time in the other three groups was shorter. The wound healing rate of adipose-derived mesenchymal stem cell conditioned medium combined with platelet-rich fibrin group was significantly higher than that in the other three groups at 3 and 7 days after treatment(P < 0.05). The adipose-derived mesenchymal stem cell conditioned medium group and platelet-rich fibrin group showed higher healing rate than the adipose-derived mesenchymal stem cell specific medium group(P < 0.05). Histological findings showed that the aggregation of white blood cells and fibroblasts, angiogenesis, granulation tissue remodeling and epithelization in the conditioned medium of adipose mesenchymal stem cells combined with the platelet-rich fibrin group were more obvious than those in the other three groups, in which the best wound healing effect was found. These results suggest that the combination of adipose tissue-derived mesenchymal stem cell conditioned medium and platelet-rich fibrin can effectively promote the repair of skin injury in mice, and the effect is better than that of conditioned medium or platelet-rich fibrin alone.
BACKGROUND: Platelet-rich fibrin can promote soft tissue healing, and soluble factors secreted by mesenchymal stem cells can promote skin regeneration, but their combination effects on skin repair are little reported. OBJECTIVE: To investigate the effect of conditioned medium of adipose tissue-derived mesenchymal stem cells combined with platelet-rich fibrin on the healing of skin wound in mice. METHODS: Full-thickness skin injury models were established in C57/BL6 mice and randomly divided into four groups, 10 mice in each group. After the modeling, each group was smeared with the specific medium of adipose tissue-derived mesenchymal stem cells, the conditioned medium of adipose tissue-derived mesenchymal stem cells, the platelet-rich fibrin, the conditioned medium of adipose tissue-derived mesenchymal stem cells combined with platelet-rich fibrin, thrice daily for 7 consecutive days. The wound size was measured at 1, 3, 7 and 10 days after treatment and the wound healing rate was calculated at 3 and 7 days after treatment. Histological analysis of wound healing was performed at 14 days after treatment. RESULTS AND CONCLUSION: Compared with the specific culture medium of adipose tissue-derived mesenchymal stem cells, the wound healing time in the other three groups was shorter. The wound healing rate of adipose-derived mesenchymal stem cell conditioned medium combined with platelet-rich fibrin group was significantly higher than that in the other three groups at 3 and 7 days after treatment(P < 0.05). The adipose-derived mesenchymal stem cell conditioned medium group and platelet-rich fibrin group showed higher healing rate than the adipose-derived mesenchymal stem cell specific medium group(P < 0.05). Histological findings showed that the aggregation of white blood cells and fibroblasts, angiogenesis, granulation tissue remodeling and epithelization in the conditioned medium of adipose mesenchymal stem cells combined with the platelet-rich fibrin group were more obvious than those in the other three groups, in which the best wound healing effect was found. These results suggest that the combination of adipose tissue-derived mesenchymal stem cell conditioned medium and platelet-rich fibrin can effectively promote the repair of skin injury in mice, and the effect is better than that of conditioned medium or platelet-rich fibrin alone.
引文
[1]Romana-Souza B, Dos Santos JS, Monte-Alto-Costa A.Caffeic acid phenethyl ester promotes wound healing of mice pressure ulcers affecting NF-κB, NOS2 and NRF2 expression.Life Sci. 2018;207:158-165.
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[14]Kouhkheil R, Fridoni M, Piryaei A, et al. The effect of combined pulsed wave low-level laser therapy and mesenchymal stem cell-conditioned medium on the healing of an infected wound with methicillin-resistant Staphylococcal aureus in diabetic rats. J Cell Biochem. 2018;119(7):5788-5797.
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[16]Wu YY, Jiao YP, Xiao LL, et al. Experimental Study on Effects of Adipose-Derived Stem Cell-Seeded Silk Fibroin Chitosan Film on Wound Healing of a Diabetic Rat Model. Ann Plast Surg. 2018;80(5):572-580.
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[19]Ge Y, Gomez NC, Adam RC, et al. Stem Cell Lineage Infidelity Drives Wound Repair and Cancer. Cell. 2017;169(4):636-650. e14.
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[21]侯晓琳,郁卫东,崔梅花,等.小鼠脂肪间充质干细胞的分离培养及肠道归巢[J].中国组织工程研究,2015,19(6):854-860.
[22]余霞,李琼,任明芬,等.小鼠脂肪源和骨髓源间充质干细胞表面标记表达的比较[J].解剖学报,2016,47(2):203-208.
[23]王晓宇,郭家辰,刘勇.脂肪间充质干细胞上清液对小鼠皮肤创伤的作用及可能机制[J].解剖科学进展,2015,21(3):263-266.
[24]楼敏铭,王宏伟,马洁.脐带间充质干细胞条件培养液联合透明质酸修复小鼠皮肤损伤[J].中国组织工程研究,2018,22(1):20-25.
[25]汪新伟,荣洁琳,蒋思静,等.富血小板纤维蛋白在治疗糖尿病兔皮肤溃疡中的实验研究[J].安徽医科大学学报, 2017,52(7):983-987.
[26]Bernasconi R, Nystr?m A. Balance and circumstance:The renin angiotensin system in wound healing and fibrosis. Cell Signal. 2018;51:34-46.
[27]Larouche J, Sheoran S, Maruyama K, et al. Immune Regulation of Skin Wound Healing:Mechanisms and Novel Therapeutic Targets. Adv Wound Care(New Rochelle). 2018;7(7):209-231.
[28]Pastar I, Wong LL, Egger AN, et al. Descriptive vs mechanistic scientific approach to study wound healing and its inhibition:Is there a value of translational research involving human subjects. Exp Dermatol. 2018;27(5):551-562.
[29]Tang D, Zhang J, Yan T, et al. FG-4592 Accelerates Cutaneous Wound Healing by Epidermal Stem Cell Activation via HIF-1αStabilization. Cell Physiol Biochem. 2018;46(6):2460-2470.
[30]Zomer HD, Trentin AG. Skin wound healing in humans and mice:Challenges in translational research. J Dermatol Sci.2018;90(1):3-12.
[31]汤沈力,谭秋雯,周宇婷,等.脂肪间充质干细胞在皮肤创伤修复中的研究进展[J].中国修复重建外科杂志,2017,31(6):745-750.
[32]Jayaraman P, Nathan P, Vasanthan P, et al. Stem cells conditioned medium:a new approach to skin wound healing management. Cell Biol Int. 2013;37(10):1122-1128.
[33]Jun EK, Zhang Q, Yoon BS, et al. Hypoxic conditioned medium from human amniotic fluid-derived mesenchymal stem cells accelerates skin wound healing through TGF-β/SMAD2 and PI3K/Akt pathways. Int J Mol Sci. 2014;15(1):605-628.
[34]Hur W, Lee HY, Min HS, et al. Regeneration of full-thickness skin defects by differentiated adipose-derived stem cells into fibroblast-like cells by fibroblast-conditioned medium. Stem Cell Res Ther. 2017;8(1):92.
[35]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.
[36]Hassan WU, Greiser U, Wang W. Role of adipose-derived stem cells in wound healing. Wound Repair Regen. 2014;22(3):313-325.
[37]Shingyochi Y, Orbay H, Mizuno H. Adipose-derived stem cells for wound repair and regeneration. Expert Opin Biol Ther.2015;15(9):1285-1292.
[38]Suh HN, Han HJ. Sonic hedgehog increases the skin wound-healing ability of mouse embryonic stem cells through the microRNA 200 family. Br J Pharmacol. 2015;172(3):815-828.
[2]Morhenn VB. The Relationship of Wound Healing with Psoriasis and Multiple Sclerosis. Adv Wound Care(New Rochelle). 2018;7(6):185-188.
[3]Ovais M, Ahmad I, Khalil AT, et al. Wound healing applications of biogenic colloidal silver and gold nanoparticles:recent trends and future prospects. Appl Microbiol Biotechnol. 2018;102(10):4305-4318.
[4]McLeod K, Walker JT, Hamilton DW. Galectin-3 regulation of wound healing and fibrotic processes:insights for chronic skin wound therapeutics. J Cell Commun Signal. 2018;12(1):281-287.
[5]徐海燕,刘斌,安然.富血小板纤维蛋白的研究进展[J].口腔医学研究,2015,31(9):939-941,944.
[6]何赤东,刘丽忠.富血小板纤维蛋白在促进软组织损伤愈合中的临床研究进展[J].南昌大学学报:医学版,2015,55(2):96-99.
[7]罗晓丁,李丹,张剑明.富血小板纤维蛋白促进组织愈合机制的探讨[J].中国口腔种植学杂志,2011,16(4):198-200.
[8]许方方,刘斌.富血小板纤维蛋白的研究及在组织修复中的应用[J].口腔医学,2015,35(8):691-693.
[9]雍志军,贾帅军,韩林章,等.富血小板纤维蛋白对兔骨髓间充质干细胞成软骨分化的影响[J].现代生物医学进展, 2014,14(8):1407-1410.
[10]宋楠,胡刚,陈锦,等.富血小板纤维蛋白促进创伤修复的研究进展[J].中国美容整形外科杂志, 2011,22(1):31-34.
[11]汪新伟,荣洁琳,蒋思静,等.富血小板纤维蛋白在治疗糖尿病兔皮肤溃疡中的实验研究[J].安徽医科大学学报, 2017,52(7):983-987.
[12]朱卫丽,薛小萍,刘衍春.富血小板纤维蛋白在临床医学中的应用研究进展[J].中国输血杂志,2016,29(5):545-548.
[13]Hamid MSA. Cost Effectiveness of a Platelet-rich Plasma Preparation Technique for Clinical Use. Wounds. 2018; 30(7):186-190.
[14]Kouhkheil R, Fridoni M, Piryaei A, et al. The effect of combined pulsed wave low-level laser therapy and mesenchymal stem cell-conditioned medium on the healing of an infected wound with methicillin-resistant Staphylococcal aureus in diabetic rats. J Cell Biochem. 2018;119(7):5788-5797.
[15]Ge Y, Fuchs E. Stretching the limits:from homeostasis to stem cell plasticity in wound healing and cancer. Nat Rev Genet. 2018;19(5):311-325.
[16]Wu YY, Jiao YP, Xiao LL, et al. Experimental Study on Effects of Adipose-Derived Stem Cell-Seeded Silk Fibroin Chitosan Film on Wound Healing of a Diabetic Rat Model. Ann Plast Surg. 2018;80(5):572-580.
[17]Tsai SH, Tsao LP, Chang SH, et al. Pigment epitheliumderived factor short peptides facilitate full-thickness cutaneous wound healing by promoting epithelial basal cell and hair follicle stem cell proliferation. Exp Ther Med. 2017;14(5):4853-4861.
[18]Park SR, Kim JW, Jun HS, et al. Stem Cell Secretome and Its Effect on Cellular Mechanisms Relevant to Wound Healing.Mol Ther. 2018;26(2):606-617.
[19]Ge Y, Gomez NC, Adam RC, et al. Stem Cell Lineage Infidelity Drives Wound Repair and Cancer. Cell. 2017;169(4):636-650. e14.
[20]周治来,徐华丽,姚舜,等.脂肪间充质干细胞移植提升脊髓损伤小鼠血小板反应蛋白4表达促进运动功能恢复[J].神经解剖学杂志,2018,34(3):371-376.
[21]侯晓琳,郁卫东,崔梅花,等.小鼠脂肪间充质干细胞的分离培养及肠道归巢[J].中国组织工程研究,2015,19(6):854-860.
[22]余霞,李琼,任明芬,等.小鼠脂肪源和骨髓源间充质干细胞表面标记表达的比较[J].解剖学报,2016,47(2):203-208.
[23]王晓宇,郭家辰,刘勇.脂肪间充质干细胞上清液对小鼠皮肤创伤的作用及可能机制[J].解剖科学进展,2015,21(3):263-266.
[24]楼敏铭,王宏伟,马洁.脐带间充质干细胞条件培养液联合透明质酸修复小鼠皮肤损伤[J].中国组织工程研究,2018,22(1):20-25.
[25]汪新伟,荣洁琳,蒋思静,等.富血小板纤维蛋白在治疗糖尿病兔皮肤溃疡中的实验研究[J].安徽医科大学学报, 2017,52(7):983-987.
[26]Bernasconi R, Nystr?m A. Balance and circumstance:The renin angiotensin system in wound healing and fibrosis. Cell Signal. 2018;51:34-46.
[27]Larouche J, Sheoran S, Maruyama K, et al. Immune Regulation of Skin Wound Healing:Mechanisms and Novel Therapeutic Targets. Adv Wound Care(New Rochelle). 2018;7(7):209-231.
[28]Pastar I, Wong LL, Egger AN, et al. Descriptive vs mechanistic scientific approach to study wound healing and its inhibition:Is there a value of translational research involving human subjects. Exp Dermatol. 2018;27(5):551-562.
[29]Tang D, Zhang J, Yan T, et al. FG-4592 Accelerates Cutaneous Wound Healing by Epidermal Stem Cell Activation via HIF-1αStabilization. Cell Physiol Biochem. 2018;46(6):2460-2470.
[30]Zomer HD, Trentin AG. Skin wound healing in humans and mice:Challenges in translational research. J Dermatol Sci.2018;90(1):3-12.
[31]汤沈力,谭秋雯,周宇婷,等.脂肪间充质干细胞在皮肤创伤修复中的研究进展[J].中国修复重建外科杂志,2017,31(6):745-750.
[32]Jayaraman P, Nathan P, Vasanthan P, et al. Stem cells conditioned medium:a new approach to skin wound healing management. Cell Biol Int. 2013;37(10):1122-1128.
[33]Jun EK, Zhang Q, Yoon BS, et al. Hypoxic conditioned medium from human amniotic fluid-derived mesenchymal stem cells accelerates skin wound healing through TGF-β/SMAD2 and PI3K/Akt pathways. Int J Mol Sci. 2014;15(1):605-628.
[34]Hur W, Lee HY, Min HS, et al. Regeneration of full-thickness skin defects by differentiated adipose-derived stem cells into fibroblast-like cells by fibroblast-conditioned medium. Stem Cell Res Ther. 2017;8(1):92.
[35]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.
[36]Hassan WU, Greiser U, Wang W. Role of adipose-derived stem cells in wound healing. Wound Repair Regen. 2014;22(3):313-325.
[37]Shingyochi Y, Orbay H, Mizuno H. Adipose-derived stem cells for wound repair and regeneration. Expert Opin Biol Ther.2015;15(9):1285-1292.
[38]Suh HN, Han HJ. Sonic hedgehog increases the skin wound-healing ability of mouse embryonic stem cells through the microRNA 200 family. Br J Pharmacol. 2015;172(3):815-828.