人羊膜上皮细胞旁分泌作用及其对糖尿病大鼠创面血管再生的影响
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摘要
目的研究人羊膜上皮细胞(human amnioticepithelial cells,hAECs)旁分泌情况及其在糖尿病皮肤损伤大鼠模型中皮肤愈合及血管再生的作用。方法采用流式细胞术对hAECs表型进行分析;ELISA方法检测hAECs培养上清中旁分泌因子含量;划痕试验检测hAECs条件培养基对人脐静脉血管内皮细胞(human umbilical veinendothelial cells,HUVECs)划痕的愈合作用,确定旁分泌作用对其迁移能力的影响;对糖尿病大鼠进行皮肤损伤造模,检测经h AECs治疗后,皮肤中CD34表达及Cyclin D1基因mRNA转录水平。结果 hAECs表面高表达间充质干细胞表面标志CD29、CD73、CD105及胚胎干细胞表面标志SSEA4,低表达间充质干细胞表面标志CD44、CD90及造血干细胞表面标志CD45、CD34、HLA-DR;可大量分泌血管内皮生长因子(vascular endothelial growth factor,VEGF),72 h时表达量显著高于24和48 h及培养基对照组(P <0. 05);HUVECs划痕后24和48 h,两种浓度条件培养基组对细胞的愈合作用均较基础培养基对照组强,差异有统计学意义(P <0. 01),且两种浓度条件培养基组细胞迁移率差异无统计学意义(P> 0. 05);hAECs治疗后糖尿病大鼠皮肤中CD34表达及Cyclin D1基因mRNA转录水平均显著高于相同取材时间条件下的未治疗组,差异有统计学意义(P <0. 05)。结论 hAECs可大量分泌VEGF,并能够通过旁分泌作用促进体外HUVECs迁移、增殖及糖尿病大鼠皮肤血管再生。
Objective To investigate the paracrine status of human amnioticepithelial cells(hAECs)and its role in wound healing and angiogenesis in diabetic skin lesions. Methods The phenotype of hAECs was analyzed by flow cytometry.The paracrine factor content in culture supernatant of hAECs was determined by ELISA. The wound healing effect of conditioned medium of h AECs on human umbilical vein endothelial cells(HUVECs)was evaluated by wound healing test.The skin lesion model of diabetic rats was established. The model rats were treated with hAECs,of which the CD34 expression and Cyclin D1 mRNA transcription levels in skin were determined. Results CD29,CD73 and CD105 as surface markers of mesenchyma stem cells(MSCs) and SSEA4 as surface marker of embryonic stem cells were highly expressed in hAECs,while CD44 and CD90 as surface markers of MSCs and CD45,CD34 and HLA-DR as surface markers of ematopoietic stem cells were low expressed. Vein endothelial growth factor(VEGF) was secreted in a large quantity,of which the expression level 72 h was significantly higher than those 24 and 48 h after culture and those in medium control group(P < 0. 05). Compared with the basal medium,the conditional media at two concentrations showed strong healing effects on HUVECs 24 and 48 h after scratch(P < 0. 01). However,the migration rates of HUVECs in conditional media at two concentrations showed no significant difference(P > 0. 05). Both the expression level of CD34 and transcription level of Cyclin D1 mRNA in skin of diabetic rats after treatment with hAECs were significantly higher than those in control group under the same condition(P < 0. 05). Conclusion The hAECs secreted VEGF in a large quantity and promoted the in vitro migration and proliferation of HUVECs as well as the skin vascular regeneration of diabetic rats through paracrine effect.
Objective To investigate the paracrine status of human amnioticepithelial cells(hAECs)and its role in wound healing and angiogenesis in diabetic skin lesions. Methods The phenotype of hAECs was analyzed by flow cytometry.The paracrine factor content in culture supernatant of hAECs was determined by ELISA. The wound healing effect of conditioned medium of h AECs on human umbilical vein endothelial cells(HUVECs)was evaluated by wound healing test.The skin lesion model of diabetic rats was established. The model rats were treated with hAECs,of which the CD34 expression and Cyclin D1 mRNA transcription levels in skin were determined. Results CD29,CD73 and CD105 as surface markers of mesenchyma stem cells(MSCs) and SSEA4 as surface marker of embryonic stem cells were highly expressed in hAECs,while CD44 and CD90 as surface markers of MSCs and CD45,CD34 and HLA-DR as surface markers of ematopoietic stem cells were low expressed. Vein endothelial growth factor(VEGF) was secreted in a large quantity,of which the expression level 72 h was significantly higher than those 24 and 48 h after culture and those in medium control group(P < 0. 05). Compared with the basal medium,the conditional media at two concentrations showed strong healing effects on HUVECs 24 and 48 h after scratch(P < 0. 01). However,the migration rates of HUVECs in conditional media at two concentrations showed no significant difference(P > 0. 05). Both the expression level of CD34 and transcription level of Cyclin D1 mRNA in skin of diabetic rats after treatment with hAECs were significantly higher than those in control group under the same condition(P < 0. 05). Conclusion The hAECs secreted VEGF in a large quantity and promoted the in vitro migration and proliferation of HUVECs as well as the skin vascular regeneration of diabetic rats through paracrine effect.
引文
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[2]EVRON A,GOLDMAN S,SHALEV E.Human amniotic epithelial cells differentiate into cells expressing germ cell specific markers when cultured in medium containing serum substitute supplement[J].Reprod Biol Endocrin,2012,10:108.
[3]LIU X Y,ZHOU Q,ZHANG X L,et al.In vitro culture and molecular characterization of human amniotic epithelial cells[J].Chin J Cell Mol Immunol,2014,30(12):1318-1321.(in Chinese)刘小勇,周清,张晓玲,等.人羊膜上皮细胞的体外培养及标志分子的检测分析[J].细胞与分子免疫学杂志,2014,30(12):1318-1321.
[4]PRATAMA G,VAGHJIANI V,TEE J Y,et al.Changes in culture expanded human amniotic epithelial cells:implications for potential therapeutic applications[J].PLoS One,2011,6(11):e26136.doi:10.1371/journal.pone.0026136.
[5]GARC魱A-CASTRO I L,GARC A-L PEZ G,VILA-GONZ LEZD,et al.Markers of pluripotency in human amniotic epithelial cells and their differentiation to progenitor of cortical neurons[J].PLoS One,2015,10(12):e0146082.doi:10.1371/journal.pone.0146082.
[6]WHITTAM A J,MAAN Z N,DUSCHER D,et al.Challenges and opportunities in drug delivery for wound healing[J].Adv Wound Care(New Rochelle),2016,5(2):79-88.
[7]GHATAK S,MAYTIN E V,MACK J A,et al.Roles of proteoglycans and glycosaminoglycans in wound healing and fibrosis[J].Int J Cell Biol,2015,2015:834893.doi:10.1155/2015/834893.
[8]CHING Y H,SUTTON T L,PIERPONT Y N,et al.The use of growth factors and other humoral agents to accelerate and enhance burn wound healing[J].Eplasty,2011,11:e41.
[9]CHEN L,XU Y,ZHAO J,et al.Conditioned medium from hypoxic bone marrow-derived mesenchymal stem cells enhances wound healing in mice[J].PLoS One,2014,9(4):e96161.doi:10.1371/journal.pone.0096161.
[10]MCDONALD C A,PAYNE N L,SUN G,et al.Immunosuppressive potential of human amnion epithelial cells in the treatment of experimental autoimmune encephalomyelitis[J].JNeuroinflammation,2015,12:112.
[11]JOHNSON K E,WILQUS T A.Vascular endothelial growth factor and angiogenesis in the regulation of cutaneous wound repair[J].Adv Wound Care(New Rochelle),2014,3(10):647-661.
[12]NUNES Q M,LI Y,SUN C,et al.Fibroblast growth factors as tissue repair and regeneration therapeutics[J].Peer J,2016,4(1):e1535.doi:10.7717/peerj.1535.
[13]ZHAO B,LIU J Q,YANG C,et al.Human amniotic epithelial cells attenuate TGF-β1-induced human dermal fibroblast transformation to myofibroblasts via TGF-β1/Smad3 pathway[J].Cytotherapy,2016,18(8):1012-1024.
[14]FU Y S,ZHANG Y,ZHANG T T,et al.The paracrine effect of human amniotic epithelial cells and their roles in skin wound healing[J].Chin J Histochem Cytochem,2017,26(2):183-190.(in Chinese)付寅生,张怡,张婷婷,等.羊膜上皮细胞旁分泌特点及其在皮肤损伤修复中的作用[J].中国组织化学与细胞化学杂志,2017,26(2):183-190.
[15]ZHU D,MULJADI R,CHAN S T,et al.Evaluating the impact of human amnion epithelial cells on angiogenesis[J].Stem Cells Int,2016,2016:4565612.doi:10.1155/2016/4565612.
[16]SALGADO A J,REIS R L,SOUSA N J,et al.Adipose tissue derived stem cells secretome:soluble factors and their roles in regenerative medicine[J].Curr Stem Cell Res Ther,2010,5(2):103-110.
[17]SONG Y S,JOO H W,PARK I H,et al.Transplanted human amniotic epithelial cells secrete paracrine proangiogenic cytokines in rat model of myocardial infarction[J].Cell Transplant,2015,24(10):2055-2064.
[18]BRYZEK A,CZEKAJ P,PLEWKA D,et al.Expression and co-expression of surface markers of pluripotency on human amniotic cells cultured in different growth media[J].Ginekol Pol,2013,84(12):1012-1024.
[19]STEED D L,TRUMPOWER C,DUFFY D,et al.Amnionderived cellular cytokine solution:a physiological combination of cytokines for wound healing[J].Eplasty,2008,8:e18.
[20]WOLBANK S,HILDNER F,REDL H,et al.Impact of human amniotic membrane preparation on release of angiogenic factors[J].J Tissue Eng Regen Med,2009,3(8):651-654.