人脐带间充质干细胞条件培养基脂质体修复大鼠皮肤创面损伤
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摘要
背景:干细胞条件培养基中含有大量有助于伤口修复的细胞分泌因子,用脂质体包裹有助于细胞因子在伤口处的缓释作用。目的:研究人脐带间充质干细胞条件培养基脂质体对大鼠皮肤创面愈合的影响。方法:采用组织块贴壁法分离培养人脐带间充质干细胞并进行相关因子检测。取20只SD大鼠(购自山西医科大学实验动物中心)用于制备直径1 cm的皮肤创面模型。薄膜分散制备条件培养基脂质体囊泡用于大鼠皮肤缺损伤口,取修复部位皮肤及周围组织进行苏木精-伊红染色病理切片观察,通过组织学评分统计分析判断其对创伤的修复情况。结果与结论:(1)人脐带间充质干细胞表面标志物CD73、CD90、CD105阳性率分别为99.7%、95.7%、99.4%,CD45、CD34阳性率分别为1.1%、0.1%,且能诱导分化为骨细胞、脂肪细胞;(2)条件培养基中血管内皮生长因子、白细胞介素8、单核细胞趋化蛋白1的质量浓度分别为558.60 ng/L、5 236.48 ng/L、2 972.55 ng/L;(3)用薄膜分散法制备得到人脐带间充质干细胞条件培养基脂质体平均粒径为262.3 nm;(4)人脐带间充质干细胞条件培养及其脂质体均可促进大鼠皮肤缺损处的毛囊、腺体发生,且表皮层加厚,组织学评分显著升高,第2天的条件培养基与条件培养基脂质体差异无显著性意义(P=0.730),其他各组之间有显著差异(P <0.05);(5)结果提示,人脐带间充质干细胞条件培养基及其脂质体可以促进大鼠缺损伤口的修复。
BACKGROUND:The stem cell conditioned medium contains a large number of cell secretory factors contributing to wound repair. Liposome encapsulation facilitates the sustained release of cytokines at the wound site.OBJECTIVE:To study the effect of liposomes prepared from the conditioned medium of human umbilical cord mesenchymal stem cells on the healing of skin wounds in rats.METHODS:Human umbilical cord mesenchymal stem cells were isolated and cultured by tissue explant method, and cytokine detection was then performed. Twenty Sprague-Dawley rats were provided by the Experimental Animal Center of Shanxi Medical University in China to make skin wound models of 1 cm in diameter. Membrane dispersion technique was used to prepare the conditioned medium liposomal vesicles used for skin wounds in the rats. The skin and surrounding tissues of the repair site were taken for hematoxylin-eosin staining. The histological scores were used to judge the repair of wounds.RESULTS AND CONCLUSION:(1) The human umbilical cord mesenchymal stem cells were positive for CD73(99.7%), CD90(95.7%),CD105(99.4%), CD45(1.1%) and CD34(0.1%). The cells could be induced to differentiate into osteocytes and adipocytes.(2) The concentrations of vascular endothelial growth factor, interleukin-8, and monocyte chemotactic protein 1 in the conditioned medium were558.60 ng/L, 5 236.48 ng/L, and 2 972.55 ng/L, respectively.(3) The average particle size of liposome in the conditioned medium prepared by membrane dispersion method was 262.3 nm. Conditional culture of human umbilical cord mesenchymal stem cells and its liposome could increase the hair follicles and glands at the site of rat skin defects, the epidermis was thickened, and the histological score was markedly increased. There was no difference between the conditioned medium and conditioned medium liposome groups at 2 days of culture(P=0.730),but significant differences existed among the other groups(P < 0.05). To conclude, the human umbilical cord mesenchymal stem cell conditioned medium and its liposomes can promote the repair of skin defects in rats.
BACKGROUND:The stem cell conditioned medium contains a large number of cell secretory factors contributing to wound repair. Liposome encapsulation facilitates the sustained release of cytokines at the wound site.OBJECTIVE:To study the effect of liposomes prepared from the conditioned medium of human umbilical cord mesenchymal stem cells on the healing of skin wounds in rats.METHODS:Human umbilical cord mesenchymal stem cells were isolated and cultured by tissue explant method, and cytokine detection was then performed. Twenty Sprague-Dawley rats were provided by the Experimental Animal Center of Shanxi Medical University in China to make skin wound models of 1 cm in diameter. Membrane dispersion technique was used to prepare the conditioned medium liposomal vesicles used for skin wounds in the rats. The skin and surrounding tissues of the repair site were taken for hematoxylin-eosin staining. The histological scores were used to judge the repair of wounds.RESULTS AND CONCLUSION:(1) The human umbilical cord mesenchymal stem cells were positive for CD73(99.7%), CD90(95.7%),CD105(99.4%), CD45(1.1%) and CD34(0.1%). The cells could be induced to differentiate into osteocytes and adipocytes.(2) The concentrations of vascular endothelial growth factor, interleukin-8, and monocyte chemotactic protein 1 in the conditioned medium were558.60 ng/L, 5 236.48 ng/L, and 2 972.55 ng/L, respectively.(3) The average particle size of liposome in the conditioned medium prepared by membrane dispersion method was 262.3 nm. Conditional culture of human umbilical cord mesenchymal stem cells and its liposome could increase the hair follicles and glands at the site of rat skin defects, the epidermis was thickened, and the histological score was markedly increased. There was no difference between the conditioned medium and conditioned medium liposome groups at 2 days of culture(P=0.730),but significant differences existed among the other groups(P < 0.05). To conclude, the human umbilical cord mesenchymal stem cell conditioned medium and its liposomes can promote the repair of skin defects in rats.
引文
[1]Dominici M,Le BK,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.
[2]Modo M,Rezaie PP,Patel S,et al.Transplantation of neural stem cells in a rat model of stroke:assessment of short-term graft survival and acute host immunological response.Brain Res.2002;958(1):70-82.
[3]Kucia M,Reca R,Miekus K,et al.Trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms:pivotal role of the SDF-1-CXCR4 axis.Stem Cells.2005;23(7):879-894.
[4]Sell S.On the stem cell origin of cancer.Am J Pathol.2010;176(6):2584-2494.
[5]Lee M J,Kim J,Lee K I,et al.Enhancement of wound healing by secretory factors of endothelial precursor cells derived from human embryonic stem cells.Cytotherapy.2011;13(2):165-178.
[6]Chen L,Tredget EE,Wu PY,et al.Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing.Plos One.2008;3(4):e1886.
[7]Kim W S,Park B S,Sung J H.Protective role of adipose-derived stem cells and their soluble factors in photoaging.Arch Dermatol Res.2009;301(5):329.
[8]Larios C,Espina M,Alsina MA,et al.Interaction of threeβ-interferon domains with liposomes and monolayers as model membranes.Biophys Chem.2004;111(2):123-133.
[9]Proksch E,Brandner JJ.The skin:an indispensable barrier.Exp Dermatol.2008;17(12):1063-1072.
[10]Gurtner GC,Werner S,Barrandon Y,et al.Wound repair and regeneration.Nature.2008;453(7193):314-321.
[11]Wang T,Fang X,Yin ZS.Endothelial progenitor cell-conditioned medium promotes angiogenesis and is neuroprotective after spinal cord injury.Neural Regen Res.2018;13(5):887-895.
[12]Monteiro N,Martins A,Rui LR,et al.Liposomes in tissue engineering and regenerative medicine.J R Soc Interface.2014;11(101):20140459.
[13]Xing Y,Wen CY,Li ST,et al.Non-viral liposome-mediated transfer of brain-derived neurotrophic factor across the blood-brain barrier.Neural Regen Res.2016;11(4):617-622.
[14]Thomas HE,Zhang Y,Stefely JA,et al.Mitochondrial complex I activity is required for maximal autophagy.Cell Rep.2018;24(9):2404-2417.
[15]Sapar ML,Ji H,Wang B,et al.Phosphatidylserine externalization results from and causes neurite degeneration in drosophila.Cell Rep.2018;24(9):2273-2286.
[16]Bozzuto G,Molinari A.Liposomes as nanomedical devices.Int J Nanomedicine.2015;2015:975-999.
[17]Yingchoncharoen P,Kalinowski DS,Richardson DR.Lipid-based drug delivery systems in cancer therapy:what is available and what is yet to come.Pharmacol Rev.2016;68(3):701-787.
[18]Irina A,Andrey E,Andrey M,et al.Umbilical cord as prospective source for mesenchymal stem cell-based therapy.Stem Cells Int.2016;.2016(3):6901286.
[19]Davies JE,Walker JT,Keating A.Concise review:wharton's jelly:the rich,but enigmatic,source of mesenchymal stromal cells.stem cells Transl Med.2017;6(7):1620.
[20]Arno AI,Amininik S,Blit PH,et al.Human Wharton’s jelly mesenchymal stem cells promote skin wound healing through paracrine signaling.Stem Cell Res Ther.2014;5(1):28.
[21]Li,ZY,Paulson AT,Gill T A.Encapsulation of bioactive salmon protein hydrolysates with chitosan-coated liposomes.J Funct Foods.2015;19:733-743.
[22]J?raholmen MW,Basnet P,Acharya G,et al.PEGylated liposomes for topical vaginal therapy improve delivery of interferon alpha.Eur J Pharm Biopharm.2017;113:132-139.
[23]Yang L,Yang W,Bi D,et al.A novel method to prepare highly encapsulated interferon-α-2b containing liposomes for intramuscular sustained release.Eur J Pharm Biopharm.2006;64(1):9-15.
[24]Boulton AJ,Vileikyte L,Ragnarsontennvall G,et al.The global burden of diabetic foot disease.Lancet.2005;366(9498):1719-1724.
[25]Dominici M,Le BK,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.
[26]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.
[27]Zhang B,Wang M,Gong A,et al.Huc MSC-exosome mediated-Wnt4 signaling is required for cutaneous wound healing.Stem Cells.2015;33(7):2158.
[28]Ueda M,Nishino Y.Cell-based cytokine therapy for skin rejuvenation.J Craniofac Surg.2010;21(6):1861-1866.
[29]王黎明.脐带间充质干细胞的分泌蛋白表达谱研究[D].合肥:安徽医科大学,2014.
[30]Arno AI,Saeid AN,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.
[31]Shin L,Peterson D A.Human mesenchymal stem cell grafts enhance normal and impaired wound healing by recruiting existing endogenous tissue stem/progenitor cells.Stem Cells Transl Med.2013;2(1):33-42.
[32]Talwadekar MD,Kale VP,Limaye LS.Placenta-derived mesenchymal stem cells possess better immunoregulatory properties compared to their cord-derived counterparts-a paired sample study.Sci Rep.2015;5:15784.
[2]Modo M,Rezaie PP,Patel S,et al.Transplantation of neural stem cells in a rat model of stroke:assessment of short-term graft survival and acute host immunological response.Brain Res.2002;958(1):70-82.
[3]Kucia M,Reca R,Miekus K,et al.Trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms:pivotal role of the SDF-1-CXCR4 axis.Stem Cells.2005;23(7):879-894.
[4]Sell S.On the stem cell origin of cancer.Am J Pathol.2010;176(6):2584-2494.
[5]Lee M J,Kim J,Lee K I,et al.Enhancement of wound healing by secretory factors of endothelial precursor cells derived from human embryonic stem cells.Cytotherapy.2011;13(2):165-178.
[6]Chen L,Tredget EE,Wu PY,et al.Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing.Plos One.2008;3(4):e1886.
[7]Kim W S,Park B S,Sung J H.Protective role of adipose-derived stem cells and their soluble factors in photoaging.Arch Dermatol Res.2009;301(5):329.
[8]Larios C,Espina M,Alsina MA,et al.Interaction of threeβ-interferon domains with liposomes and monolayers as model membranes.Biophys Chem.2004;111(2):123-133.
[9]Proksch E,Brandner JJ.The skin:an indispensable barrier.Exp Dermatol.2008;17(12):1063-1072.
[10]Gurtner GC,Werner S,Barrandon Y,et al.Wound repair and regeneration.Nature.2008;453(7193):314-321.
[11]Wang T,Fang X,Yin ZS.Endothelial progenitor cell-conditioned medium promotes angiogenesis and is neuroprotective after spinal cord injury.Neural Regen Res.2018;13(5):887-895.
[12]Monteiro N,Martins A,Rui LR,et al.Liposomes in tissue engineering and regenerative medicine.J R Soc Interface.2014;11(101):20140459.
[13]Xing Y,Wen CY,Li ST,et al.Non-viral liposome-mediated transfer of brain-derived neurotrophic factor across the blood-brain barrier.Neural Regen Res.2016;11(4):617-622.
[14]Thomas HE,Zhang Y,Stefely JA,et al.Mitochondrial complex I activity is required for maximal autophagy.Cell Rep.2018;24(9):2404-2417.
[15]Sapar ML,Ji H,Wang B,et al.Phosphatidylserine externalization results from and causes neurite degeneration in drosophila.Cell Rep.2018;24(9):2273-2286.
[16]Bozzuto G,Molinari A.Liposomes as nanomedical devices.Int J Nanomedicine.2015;2015:975-999.
[17]Yingchoncharoen P,Kalinowski DS,Richardson DR.Lipid-based drug delivery systems in cancer therapy:what is available and what is yet to come.Pharmacol Rev.2016;68(3):701-787.
[18]Irina A,Andrey E,Andrey M,et al.Umbilical cord as prospective source for mesenchymal stem cell-based therapy.Stem Cells Int.2016;.2016(3):6901286.
[19]Davies JE,Walker JT,Keating A.Concise review:wharton's jelly:the rich,but enigmatic,source of mesenchymal stromal cells.stem cells Transl Med.2017;6(7):1620.
[20]Arno AI,Amininik S,Blit PH,et al.Human Wharton’s jelly mesenchymal stem cells promote skin wound healing through paracrine signaling.Stem Cell Res Ther.2014;5(1):28.
[21]Li,ZY,Paulson AT,Gill T A.Encapsulation of bioactive salmon protein hydrolysates with chitosan-coated liposomes.J Funct Foods.2015;19:733-743.
[22]J?raholmen MW,Basnet P,Acharya G,et al.PEGylated liposomes for topical vaginal therapy improve delivery of interferon alpha.Eur J Pharm Biopharm.2017;113:132-139.
[23]Yang L,Yang W,Bi D,et al.A novel method to prepare highly encapsulated interferon-α-2b containing liposomes for intramuscular sustained release.Eur J Pharm Biopharm.2006;64(1):9-15.
[24]Boulton AJ,Vileikyte L,Ragnarsontennvall G,et al.The global burden of diabetic foot disease.Lancet.2005;366(9498):1719-1724.
[25]Dominici M,Le BK,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.
[26]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.
[27]Zhang B,Wang M,Gong A,et al.Huc MSC-exosome mediated-Wnt4 signaling is required for cutaneous wound healing.Stem Cells.2015;33(7):2158.
[28]Ueda M,Nishino Y.Cell-based cytokine therapy for skin rejuvenation.J Craniofac Surg.2010;21(6):1861-1866.
[29]王黎明.脐带间充质干细胞的分泌蛋白表达谱研究[D].合肥:安徽医科大学,2014.
[30]Arno AI,Saeid AN,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.
[31]Shin L,Peterson D A.Human mesenchymal stem cell grafts enhance normal and impaired wound healing by recruiting existing endogenous tissue stem/progenitor cells.Stem Cells Transl Med.2013;2(1):33-42.
[32]Talwadekar MD,Kale VP,Limaye LS.Placenta-derived mesenchymal stem cells possess better immunoregulatory properties compared to their cord-derived counterparts-a paired sample study.Sci Rep.2015;5:15784.
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