分离与鉴定人脂肪间充质干细胞来源的外泌体
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摘要
背景:脂肪间充质干细胞是重要的种子细胞之一,可通过较简单的方法大量提取,但其储存条件较苛刻,运输不便,临床应用较困难。外泌体可由脂肪间充质干细胞分泌,其结构稳定,不易分解,为脂肪间充质干细胞在临床应用提供了新的可能性。目的:提取人脂肪间充质干细胞,鉴定脂肪间充质干细胞,诱导脂肪间充质干细胞多向分化,提取脂肪间充质干细胞来源的外泌体,鉴定脂肪间充质干细胞来源的外泌体。方法:收集青岛大学附属医院医学美容中心的正常成年女性行吸脂术后腹部浅层皮下脂肪组织,用酶消化法分离提取脂肪间充质干细胞进行原代培养,用细胞贴壁法纯化,胰酶消化传代。选取第3代细胞分别行流式细胞学鉴定,成脂肪细胞诱导与鉴定;成骨细胞诱导与鉴定。取第3-6代脂肪间充质干细胞,收集细胞上清液,差速离心法提取外泌体,BCA法测定蛋白浓度,透射电镜观察外泌体形态,粒度仪测外泌体直径分布,免疫磁珠法和流式细胞学鉴定。结果与结论:①脂肪间充质干细胞形态为长梭形,形似成纤维细胞,排列紧密时呈巢状;②流式细胞学检测显示CD29,CD44,CD90及CD105均为阳性表达,CD34和CD45为阴性表达;③成骨细胞诱导至第9天碱性磷酸酶染色为蓝紫色,诱导至21 d时,茜素红S染色可见细胞外基质内出现红色钙结节;④成脂肪细胞诱导14 d后油红O染色呈橘红色;⑤透射电镜显示外泌体结构为杯状,直径为(81.225±22.226) nm,有膜结构。蛋白浓度约为1.5 g/L,直径分布集中于70-100 nm,外泌体流式细胞学显示CD9,CD29,CD44,CD63,CD90和CD105为阳性表达,CD34和CD45为阴性表达;⑥结果证实,人脂肪间充质干细胞来源的外泌体能通过超速离心法顺利提取,并通过透射电子显微镜、免疫磁珠法和流式细胞学检测相结合的方法成功鉴定。
BACKGROUND:Adipose-derived mesenchymal stem cells as one of the important seed cells can be extracted in large quantities by simple methods.However,clinical application of the cells is limited by rigorous storage conditions and inconvenient transportation.Exosomes can be secreted by adipose-derived mesenchymal stem cells,whose structure is stable and difficult to decompose,providing new possibilities for clinical application of adipose-derived mesenchymal stem cells.OBJECTIVE:To isolate and identify human adipose-derived mesenchymal stem cells and induce multidirectional differentiation of the cells as well as to isolate and identify the exosomes from adipose-derived mesenchymal stem cells.METHODS:Adipose-derived mesenchymal stem cells were isolated from superficial abdominal subcutaneous adipose tissue of normal adult women after liposuction.The cells were purified by cell adherence method.The cells were digested and passaged by trypsin.The third generation cells were identified by flow cytometry,followed by adipocyte induction and identification as well as osteoblast induction and identification.Adipose-derived mesenchymal stem cells of the 3~(rd) to 6~(th) generations were collected.The exosomes were extracted from cell supernatant by differential centrifugation.The protein concentration was measured by BCA.The morphology of the exosomes was observed by transmission electron microscopy.The diameter distribution of the exosomes was measured by particle size analyzer.The exosomes were identified by immunomagnetic beads and flow cytometry.RESULTS AND CONCLUSION:Adipose-derived mesenchymal stem cells were long spindle-shaped,fibroblasts-like,when arranged tightly.Flow cytometry showed that adipose-derived mesenchymal stem cells were positive for CD29,CD44,CD90 and CD105,but negative for CD34 and CD45.Alkaline phosphatase staining was blue-purple on the 9~(th) day after induction of osteoblasts.Alizarin red S staining showed red calcium nodules in the extracellular matrix on the 21~(st) day after induction.Oil red O staining was orange on the 14~(th) day after induction of adipocytes.Under the transmission electron microscope the exosome was cup-shaped with a diameter of(81.225±22.226)nm and had a membrane structure.The protein concentration was about 1.5 g/L and the diameter distribution ws concentrated in 70-100 nm.Flow cytometry showed that the exosomes were positive for CD9,CD29,CD44,CD63,CD90 and CD105,but negative for CD34 and CD45.To conclude,the exosomes derived from adipose-derived mesenchymal stem cells can be successfully extracted by ultracentrifugation and identified by transmission electron microscopy,immunomagnetic beads and flow cytometry.
BACKGROUND:Adipose-derived mesenchymal stem cells as one of the important seed cells can be extracted in large quantities by simple methods.However,clinical application of the cells is limited by rigorous storage conditions and inconvenient transportation.Exosomes can be secreted by adipose-derived mesenchymal stem cells,whose structure is stable and difficult to decompose,providing new possibilities for clinical application of adipose-derived mesenchymal stem cells.OBJECTIVE:To isolate and identify human adipose-derived mesenchymal stem cells and induce multidirectional differentiation of the cells as well as to isolate and identify the exosomes from adipose-derived mesenchymal stem cells.METHODS:Adipose-derived mesenchymal stem cells were isolated from superficial abdominal subcutaneous adipose tissue of normal adult women after liposuction.The cells were purified by cell adherence method.The cells were digested and passaged by trypsin.The third generation cells were identified by flow cytometry,followed by adipocyte induction and identification as well as osteoblast induction and identification.Adipose-derived mesenchymal stem cells of the 3~(rd) to 6~(th) generations were collected.The exosomes were extracted from cell supernatant by differential centrifugation.The protein concentration was measured by BCA.The morphology of the exosomes was observed by transmission electron microscopy.The diameter distribution of the exosomes was measured by particle size analyzer.The exosomes were identified by immunomagnetic beads and flow cytometry.RESULTS AND CONCLUSION:Adipose-derived mesenchymal stem cells were long spindle-shaped,fibroblasts-like,when arranged tightly.Flow cytometry showed that adipose-derived mesenchymal stem cells were positive for CD29,CD44,CD90 and CD105,but negative for CD34 and CD45.Alkaline phosphatase staining was blue-purple on the 9~(th) day after induction of osteoblasts.Alizarin red S staining showed red calcium nodules in the extracellular matrix on the 21~(st) day after induction.Oil red O staining was orange on the 14~(th) day after induction of adipocytes.Under the transmission electron microscope the exosome was cup-shaped with a diameter of(81.225±22.226)nm and had a membrane structure.The protein concentration was about 1.5 g/L and the diameter distribution ws concentrated in 70-100 nm.Flow cytometry showed that the exosomes were positive for CD9,CD29,CD44,CD63,CD90 and CD105,but negative for CD34 and CD45.To conclude,the exosomes derived from adipose-derived mesenchymal stem cells can be successfully extracted by ultracentrifugation and identified by transmission electron microscopy,immunomagnetic beads and flow cytometry.
引文
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[2]Liao HT,Chen CT.Osteogenic potential:comparison between bone marrow and adipose-derived mesenchymal stem cells.World J Stem Cells.2014;6(3):288.
[3]De Ugarte DA,Ashjian PH,Elbarbary A,et al.Future of fat as raw material for tissue regeneration.Ann Plas Surg.2003;50(2):215-219.
[4]Zhang J,Li S,Li L,et al.Exosome and exosomal microrna:trafficking,sorting,and function.Genom Proteom Bioinf.2015;13(1):17
[5]van der Pol E,Boing AN,Harrison P,et al.Classification,functions,and clinical relevance of extracellular vesicles.Pharmacol Rev.2012;64(3):676.
[6]Richard JS,Hina K,Suresh M.ExoCarta as a resource for exosomal research.J Extracell Vesicles.2012.doi:10.3402/jev.v1i0.18374.
[7]Zuk PA,Zhu M,Ashjian P.human adipose tissue is a source of multipotent stem cells.Mol Biol Cell.2002;13(12):4279-4295.
[8]De Ugarte DA,Alfonso Z,Zuk PA,et al.Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow.Immunol Lett.2003;89(2-3):267.
[9]Bunnell BA,Flaat M,Gagliardi C,et al.Adipose-derived stem cells:isolation,expansion and differentiation.Methods.2008;45(2):115.
[10]Arrigoni E,Lopa S,de Girolamo L,et al.Isolation,characterization and osteogenic differentiation of adipose-derived stem cells:from small to large animal models.Cell Tissue Res.2009;338(3):401
[11]Palumbo P,Lombardi F,Siragusa G,et al.Methods of isolation,characterization and expansion of human adipose-derived stem cells(ASCs):an overview.Int J Mol Sci.2018;19(7):1897.
[12]Bliley JM,Argenta A,Satish L,et al.Administration of adipose-derived stem cells enhances vascularity,induces collagen deposition,and dermal adipogenesis in burn wounds.Burns.2016;42(6):1212.
[13]Liew L,Ong H,Dilley R.Isolation and culture of adipose-derived stromal cells from subcutaneous fat.Methods Mol Biol.2017;1627:193.
[14]Riis S.Critical steps in the isolation and expansion of adipose-derived stem cells for translational therapy.Expert Rev Mol Med.2015;17:e11.
[15]Jaiswal N,Haynesworth SE,Caplan AI,et al.Osteogenic differentiation of purified,culture-expanded human mesenchymal stem cells in vitro.J Cell Biochem.1997;64(2):295.
[16]Baglioni S,Francalanci M,Squecco R,et al.Characterization of human adult stem-cell populations isolated from visceral and subcutaneous adipose tissue.FASEB J.2009;23(10):3494.
[17]Farshid G,Kristen EL,Hani AA,et al.Clonal analysis of the differentiation potential of human adipose-derived adult stem cells.J Cell Physiol.2006;206(1):229-237.
[18]Jason W,Robert S,Mason MD,et al.Cancer exosomes trigger fibroblast to myofibroblast differentiation.Cancer Res.2010;70(23):9621
[19]方硕.干细胞来源的外泌体对成纤维细胞向肌成纤维细胞分化的调控作用及其机制研究[D].上海:第二军医大学,2016.
[20]王伟伟.HiPSC-MSCs来源Exosome促进部分肝切除后肝再生修复作用的研究[D].苏州:苏州大学,2016.
[21]王娟.脂肪干细胞来源的外来体促进皮肤创伤愈合的研究[D].武汉:华中科技大学,2016.
[22]Ruenn Chai L,Fatih A,Soon Sim T,et al.Derivation and characterization of human fetal MSCs:an alternative cell source for large-scale production of cardioprotective microparticles.J Mol Cell Cardiol.2010,48(6):1215-1224.
[23]Zuk PA,Zhu M,Mizuno H,et al.Multilineage cells from human adipose tissue:implications for cell-based therapies.Tissue Eng.2001;7(2):211-218.
[24]Toyserkani NM,Christensen ML,Sheikh SP,et al.Adipose-derived stem cells:new treatment for wound healing?Ann Plas Surg.2015;75(1):117-123.
[25]John KF,Isabella W,Zeni A,et al.Fat tissue:an underappreciated source of stem cells for biotechnology.Trends Biotechnol.2006;24(4):150-154.
[26]Zhu Y,Liu T,Song K,et al.Adipose-derived stem cell:a better stem cell than BMSC.Cell Biochem Funct.2008;26(6):664.
[27]Yoshimura K,Shigeura T,Matsumoto D,et al.Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates.J Cell Physiol.2006;208(1):64.
[28]McIntosh K,Zvonic S,Garrett S,et al.The immunogenicity of human adipose-derived cells:temporal changes in vitro.Stem Cells.2006;24(5):1246-1253.
[29]Aust L,Devlin B,Foster SJ,et al.Yield of human adiposederived adult stem cells from liposuction aspirates.Cytotherapy.2004;6(1):7.
[30]Di Taranto G,Cicione C,Visconti G,et al.Qualitative and quantitative differences of adipose-derived stromal cells from superficial and deep subcutaneous lipoaspirates:a matter of fat.Cytotherapy.2015;(8):1076
[31]毛曦媛,程辰,李华,等.不同部位脂肪组织来源干细胞的特性[J].中华整形外科杂志,2015,31(3):234.
[32]Mitchell JB,McIntosh K,Zvonic S,et al.Immunophenotype of human adipose-derived cells:temporal changes in stromal-associated and stem cell-associated markers.Stem cells(Dayton,Ohio).2006;24(2):376-385.
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