自噬在hAMSCs促进血管新生作用的初探

摘要/Abstract

摘要： 目的：通过构建人羊膜间充质干细胞（hAMSCs）与人脐静脉内皮细胞（hUVECs）3D共培养体系，探讨自噬与血管新生的相关性。方法：使用蛋白酶和胶原酶消化法及胶原酶消化法提取hAMSCs和hUVECs。通过流式细胞仪、免疫荧光及多向分化实验鉴定hAMSCs和hUVECs。通过3D成管实验观察0、3、6、12、24、48、72 h hAMSCs-hUVECs（共培养）组及hUVECs（单培养）组中hUVECs出芽的长度及面积，检测各时间点中反应自噬水平的蛋白ATG5、Beclin-1、LC3Ⅱ表达水平。结果：流式细胞仪检测发现，hAMSCs中间充质细胞表面分子标志呈阳性表达，造血干细胞及血管细胞相关的表面分子标志呈阴性表达；hUVECs中CD34呈阳性表达，CD45呈阴性表达。免疫荧光检测发现，hUVECs细胞膜上表达CD31，胞浆中表达抗血管性血友病因子（vWF）。多向分化实验证实hAMSCs具有向成骨细胞、成脂细胞及成软骨细胞分化的潜能。3D成管实验中共培养组在12 h以后出芽的长度及面积均高于单培养组，共培养组ATG5表达水平在3、6 h高于单培养组，共培养组Beclin-1表达水平在0、3、6 h高于单培养组，共培养组LC3Ⅱ表达水平在0、3、6、12 h高于单培养组。结论：本研究发现hAMSCs可以促进血管新生，并证明其促进作用与共培养体系建立早期的胞内自噬水平增高密切相关。

Abstract: The purpose of this study was to discuss the correlation of autophagy and angiogenic capacity in co-culture using human amniotic membrane-derived mesenchymal stem cells (hAMSCs) with human umbilical vein endothelial cells (hUVECs) in 3D gels by comparing to hUVECs-monoculture. Methods：To isolate hAMSCs and hUVECs, Amnion were incubated with dispase and collagenase, while umbilical veins were digested with collagenase type 1. Identification of hAMSCs and hUVECs through Flow Cytometry , immunofluorescence staining and Multilineage differentiation. The length of sprouting and average vessel area were surveyed in hUVEC-hAMSC co-culture and hUVECs-monoculture at 0, 3, 6, 12, 24, 48, 72h. ATG5, Beclin-1 and LC3Ⅱ was analyzed in hUVEC-hAMSC co-culture and hUVECs-monoculture by western blotting a 0, 3, 6, 12, 24, 48, 72h. Results：hAMSCs were positive for MSC markers, while negative for hematopoietic and vascular cellrelated markers. hUVECs were positive for CD34 and negative for CD45. hUVECs expressed CD31 on membrane surface and vWF in cytoplasm. The length of sprouting and average vessel area in hUVEC-hAMSC co-culture were more than those in hUVECs-monoculture after 12 h. Expression of ATG5 in hUVEC-hAMSC co-culture increased significantly in hUVECs –monoculture at 3 and 6 h, while Beclin 1 and LC3Ⅱ rose remarkably in hUVEC-hAMSC co-culture before 6 h and 12h respectively. Conclusions：In conclusion, this study discovered that hAMSCs can promote angiogenic capacity and demonstrated that the capacity of promote angiogenic had intimate connection with elevated level of intracellular autophagy at early co-culture system.

中图分类号:

R782.13

梁易江飞沈铭陈宁. 自噬在hAMSCs促进血管新生作用的初探[J]. , 2016, 7(1): 12-18.

参考文献

[1]Sun Q, Chen RR, Shen Y, et al.Sustained vascular endothelial growth factor delivery enhances angiogenesis and perfusion in ischemic hind limb[J].Pharm Res, 2005, 22(7):1110-1116
[2]Zisch AH, Lutolf MP, Ehrbar M, et al.Cell-demanded release of VEGF from synthetic, biointeractive cell ingrowth matrices for vascularized tissue growth[J].FASEB J, 2003, 17(15):2260-2262
[3]Sun Q, Silva EA, Wang A, et al.Sustained release of multiple growth factors from injectable polymeric system as a novel therapeutic approach towards angiogenesis[J].Pharm Res, 2010, 27(2):264-271
[4]Ren L, Ma D, Liu B, et al.Preparation of three-dimensional vascularized MSC cell sheet constructs for tissue regeneration[J].Biomed Res Int, 2014, 2014:301279-
[5]Ghajar CM, Kachgal S, Kniazeva E, et al.Mesenchymal cells stimulate capillary morphogenesis via distinct proteolytic mechanisms[J].Exp Cell Res, 2010, 316(5):813-825
[6]Kachgal S, Putnam AJ.Mesenchymal stem cells from adipose and bone marrow promote angiogenesis via distinct cytokine and protease mechanisms[J].Angiogenesis, 2011, 14(1):47-59
[7]Merfeld-Clauss S, Gollahalli N, March KL, et al.Adipose tissue progenitor cells directly interact with endothelial cells to induce vascular network formation[J].Tissue Eng Part A, 2010, 16(9):2953-2966
[8]Chen X, Aledia AS, Popson SA, et al.Rapid anastomosis of endothelial progenitor cell-derived vessels with host vasculature is promoted by a high density of cotransplanted fibroblasts[J].Tissue Eng Part A, 2010, 16(2):585-594
[9]Shepherd BR, Jay SM, Saltzman WM, et al.Human aortic smooth muscle cells promote arteriole formation by coengrafted endothelial cells[J].Tissue Eng Part A, 2009, 15(1):165-173
[10]Bergers G, Song S.The role of pericytes in blood-vessel formation and maintenance[J].Neuro Oncol, 2005, 7(4):452-464
[11]Soncini M, Vertua E, Gibelli L, et al.Isolation and characterization of mesenchymal cells from human fetal membranes[J].J Tissue Eng Regen Med, 2007, 1(4):296-305
[12]Ilancheran S, Moodley Y, Manuelpillai U.Human fetal membranes: a source of stem cells for tissue regeneration and repair?[J].Placenta, 2009, 30(1):2-10
[13]de Girolamo L, Lucarelli E, Alessandri G, et al.Mesenchymal stem/stromal cells: a new ''cells as drugs'' paradigm. Efficacy and critical aspects in cell therapy[J].Curr Pharm Des, 2013, 19(13):2459-2473
[14]Alviano F, Fossati V, Marchionni C, et al.Term Amniotic membrane is a high throughput source for multipotent Mesenchymal Stem Cells with the ability to differentiate into endothelial cells in vitro[J].BMC Dev Biol, 2007, 7:11-
[15]Kim SW, Zhang HZ, Kim CE,et al.Amniotic mesenchymal stem cells have robust angiogenic properties and are effective in treating hindlimb ischaemia[J].Cardiovasc Res, 2012, 93(3):525-534
[16]Li Y, Guo L, Ahn HS, et al.Amniotic mesenchymal stem cells display neurovascular tropism and aid in the recovery of injured peripheral nerves[J].J Cell Mol Med, 2014, 18(6):1028-1034
[17]K?nig J, Huppertz B, Desoye G,et al.Amnion-derived mesenchymal stromal cells show angiogenic properties but resist differentiation into mature endothelial cells[J].Stem Cells Dev, 2012, 21(8):1309-1320
[18]Salemi S, Yousefi S, Constantinescu MA,et al.Autophagy is required for self-renewal and differentiation of adult human stem cells[J].Cell Res, 2012, 22(2):432-435
[19]Mizushima N, Levine B.Autophagy in mammalian development and differentiation[J].Nat Cell Biol, 2010, 12(9):823-830
[20]Mathew R, White E.Autophagy, stress, and cancer metabolism: what doesn't kill you makes you stronger[J].Cold Spring Harb Symp Quant Biol, 2011, 76:389-396
[21]Shintani T, Klionsky DJ.Autophagy in health and disease: a double-edged sword[J].Science, 2004, 306(5698):990-995
[22]Levine B, Kroemer G.Autophagy in the pathogenesis of disease[J].Cell, 2008, 132(1):27-42
[23]Rubinsztein DC.The roles of intracellular protein-degradation pathways in neurodegeneration[J].Nature, 2006, 443(7113):780-786
[24]Cecconi F, Levine B.The role of autophagy in mammalian development: cell makeover rather than cell death[J].Dev Cell, 2008, 15(3):344-357
[25]Deretic V, Levine B.Autophagy,immunity,and microbial adaptations[J].Cell Host Microbe, 2009, 5(6):527-549
[26]Mizushima N, Klionsky DJ.Protein turnover via autophagy: implications for metabolism[J].Annu Rev Nutr, 2007, 27:19-40
[27]Mizushima N, Levine B, Cuervo AM, et al.Autophagy fights disease through cellular self-digestion[J].Nature, 2008, 451(7182):1069-1075
[28]Virgin HW, Levine B.Autophagy genes in immunity[J].Nat Immunol, 2009, 10(5):461-470
[29]White E, Karp C, Strohecker AM,et al.Role of autophagy in suppression of inflammation and cancer[J].Curr Opin Cell Biol, 2010, 22(2):212-217
[30]Ghajar CM, Blevins KS, Hughes CC, et al.Mesenchymal stem cells enhance angiogenesis in mechanically viable prevascularized tissues via early matrix metalloproteinase upregulation[J].Tissue Eng, 2006, 12(10):2875-2888
[31]Cai X, Lin Y, Friedrich CC, et al.Bone marrow derived pluripotent cells are pericytes which contribute to vascularization[J].Stem Cell Rev, 2009, 5(4):437-445
[32]Rao RR, Peterson AW, Ceccarelli J, et al.Matrix composition regulates three-dimensional network formation by endothelial cells and mesenchymal stem cells in collagenfibrin materials[J].Angiogenesis, 2012, 15(2):253-264
[33]Nakahara H, Bruder SP, Haynesworth SE, et al.Bone and cartilage formation in diffusion chambers by subcultured cells derived from the periosteum[J].Bone, 1990, 11(3):181-188
[34]Pittenger MF, Mackay AM, Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells[J].Science, 1999, 284(5411):143-147
[35]Rodriguez AM, Elabd C, Amri EZ,et al.The human adipose tissue is a source of multipotent stem cells[J].Biochimie, 2005, 87(1):125-128
[36]Young HE, Steele TA, Bray RA,et al.Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal,adult,and geriatric donors[J].Anat Rec, 2001, 264(1):51-62
[37]Zvaifler NJ, Marinova-Mutafchieva L, Adams G, et al.Mesenchymal precursor cells in the blood of normal individuals[J].Arthritis Res, 2000, 2(6):477-488
[38]Harichandan A, Bühring HJ.Prospective isolation of human MSC[J].Best Pract Res Clin Haematol, 2011, 24(1):25-36
[39]Isern J, Méndez-Ferrer S.Stem cell interactions in a bone marrow niche[J].Curr Osteoporos Rep, 2011, 9(4):210-218
[40]Kmiecik G, Niklińska W, Ku? P,et al.Fetal membranes as a source of stem cells[J].Adv Med Sci, 2013, 58(2):185-195
[41]Zhang Y, Li C, Jiang X, et al.Human placenta-derived mesenchymal progenitor cells support culture expansion of long-term culture-initiating cells from cord blood CD34+ cells[J].Exp Hematol, 2004, 32(7):657-664
[42]Girma JP, Meyer D, Verweij CL,et al.Structure-function relationship of human von Willebrand factor[J].Blood, 1987, 70(3):605-611
[43]Albelda SM, Muller WA, Buck CA,et al.Molecular and cellular properties of PECAM-1 (endoCAM/CD31): a novel vascular cell-cell adhesion molecule[J].J Cell Biol, 1991, 114(5):1059-1068
[44]Fariha MM, Chua KH, Tan GC, et al.Human chorion-derived stem cells: changes in stem cell properties during serial passage[J].Cytotherapy, 2011, 13(5):582-593
[45]Wan Safwani WK, Makpol S, Sathapan S, et al.The changes of stemness biomarkers in human adipose-derived stem cells during long-term manipulation[J].Biotechnol Appl Biochem, 2011, 58(4):261-270
[46]Wu Y, Chen L, Scott PG, et al.Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis[J].Stem Cells, 2007, 25(10):2648-2659
[47]Geisler S, Holmstr?m KM, Skujat D,et al.PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1[J].Nat Cell Biol, 2010, 12(2):119-131
[48]Separovic D, Kelekar A, Nayak AK,et al.Increased ceramide accumulation correlates with downregulation of the autophagy protein ATG-7 in MCF-7 cells sensitized to photodamage[J].Arch Biochem Biophys, 2010, 494(1):101-105
[49]Shen HM, Codogno P.Autophagy is a survival force via suppression of necrotic cell death[J].Exp Cell Res., 2012, 318(11):1304-1308
[50]Otomo C, Metlagel Z, Takaesu G,et al.Structure of the human ATG12~ATG5 conjugate required for LC3 lipidation in autophagy[J].Nat Struct Mol Biol, 2013, 20(1):59-66
[51]Gottlieb RA, Mentzer RM.Autophagy during cardiac stress: joys and frustrations of autophagy[J].Annu Rev Physiol, 2010, 72:45-59
[52]Itakura E, Mizushima N.Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins[J].Autophagy, 2010, 6(6):764-776
[53]Rubinsztein DC, Gestwicki JE, Murphy LO,et al.Potential therapeutic applications of autophagy[J].Nat Rev Drug Discov, 2007, 6(4):304-312
[54]Tan VP, Miyamoto S.Nutrient-sensing mTORC1: Integration of metabolic and autophagic signals[J].J Mol Cell Cardiol, 2016, :-
[55]Bhuiyan MS, Pattison JS, Osinska H,et al.Enhanced autophagy ameliorates cardiac proteinopathy[J].J Clin Invest, 2013, 123(12):5284-5297
[56]Kanamori H, Takemura G, Goto K,et al.Autophagy limits acute myocardial infarction induced by permanent coronary artery occlusion[J].Am J Physiol Heart Circ Physiol, 2011, 300(6):H2261-H2271
[57]Lum JJ, Bauer DE, Kong M,et al.Growth factor regulation of autophagy and cell survival in the absence of apoptosis[J].Cell, 2005, 120(2):237-248
[58]Matsui Y, Takagi H, Qu X,et al.Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy[J].Circ Res, 2007, 100(6):914-922
[59]Nakai A, Yamaguchi O, Takeda T,et al.The role of autophagy in cardiomyocytes in the basal state and in response to hemodynamic stress[J].Nat Med, 2007, 13(5):619-624
[60]Rabinowitz JD, White E.Autophagy and metabolism[J].Science, 2010, 330(6009):1344-1348
[61]Takagi H, Matsui Y, Hirotani S, et al.AMPK mediates autophagy during myocardial ischemia in vivo[J].Autophagy, 2007, 3(4):405-407
[62]Yan L, Vatner DE, Kim SJ, et al.Autophagy in chronically ischemic myocardium[J].Proc Natl Acad Sci U S A, 2005, 102(39):13807-13812
[63]Rouschop KM, Wouters BG.Regulation of autophagy through multiple independent hypoxic signaling pathways[J].Curr Mol Med, 2009, 9(4):417-424
[64]Mizushima N, Levine B, Cuervo AM, et al.Autophagy fights disease through cellular self-digestion[J].Nature, 2008, 451(7182):1069-1075