骨髓间充质干细胞在Matrigel凝胶支架上的生长与变化
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摘要
背景:研究表明,Matrigel作为一种细胞外基质复合物加入到细胞培养体系中,对细胞的增殖、分化、胶原分泌具有促进作用。目的:建立Matrigel复合骨髓间充质干细胞的三维培养模型,观察Matrigel三维培养条件下骨髓间充质干细胞的形态、增殖及存活情况。方法:全骨髓贴壁法分离培养骨髓间充质干细胞,进行成骨、成脂诱导鉴定;CCK-8测定第3代骨髓间充质干细胞的生长曲线;选取生长状态良好的第2代骨髓间充质干细胞,与Matrigel混合形成细胞凝胶复合物,采用相差显微镜和苏木精-伊红染色观察骨髓间充质干细胞的形态及增殖情况,Live/Dead染色评价细胞活性。结果与结论:(1)全骨髓贴壁法分离培养的比格犬骨髓间充质干细胞具备向成骨、成脂细胞分化的能力;(2)骨髓间充质干细胞的生长曲线呈S形,符合正常细胞的生长特性;(3)相差显微镜下可见骨髓间充质干细胞在Matrigel中逐渐伸长相互交联,呈三维网状生长,增殖状态良好,随着时间延长(7 d后),基质胶逐渐变软塌陷,14 d时仍可见少部分细胞网状交联生长;苏木精-伊红染色可见第4天时细胞胞质较大,第7天时细胞细长、相互交联;(4)三维培养第1,4,7天,骨髓间充质干细胞在Matrigel中活性百分比分别为92.57%,95.54%,97.37%,呈逐渐升高趋势;(5)结果表明,骨髓间充质干细胞在Matrigel中的增殖能力较强,活性较高。
BACKGROUND: Matrigel as an extracellular matrix complex can facilitate cell proliferation, differentiation and collagen secretion in a cell culture system. OBJECTIVE: To establish a three-dimensional culture model of Matrigel combined with bone marrow mesenchymal stem cells(BMSCs) and to observe the morphology, proliferation and survival of BMSCs in the Matrigel three-dimensional culture model. METHODS: BMSCs were isolated and cultured by the whole bone marrow adherent method, followed by osteogenic and adipogenic induction and identification. The growth curve of passage 3 BMSCs was determined through the CCK-8 experiment. Passage 2 BMSCs were combined with Matrigel, and the morphology and proliferation of BMSCs were observed by hematoxylin-eosin staining under a phase contrast microscope. The viability of the cells was evaluated by the Live/Dead staining. RESULTS AND CONCLUSION:(1) BMSCs cultured by the whole bone marrow adherent method had the ability to differentiate into osteoblasts and adipocytes.(2) BMSCs exhibited an "S"-shaped growth curve, which was consistent with the growth characteristics of normal cells.(3) Under the phase contrast microscope, BMSCs cultured by the Matrigel model were extended and interconnected in a three-dimensional network growth state with good proliferation. The Matrigel gradually became soft and collapsed over time(7 days) and a few cells were still in a network growth after 14 days. Hematoxylin-eosin staining results showed that the cell cytoplasm was larger at 4 days and the cells became thinned and mutually cross-linked at 7 days.(4) The active percentage of BMSCs in the Matrigel model was 92.57%, 95.54% and 97.37% at 1, 4, 7 days of culture, respectively. To conclude, BMSCs cultured on the Matrigel has good proliferation and high viability.
BACKGROUND: Matrigel as an extracellular matrix complex can facilitate cell proliferation, differentiation and collagen secretion in a cell culture system. OBJECTIVE: To establish a three-dimensional culture model of Matrigel combined with bone marrow mesenchymal stem cells(BMSCs) and to observe the morphology, proliferation and survival of BMSCs in the Matrigel three-dimensional culture model. METHODS: BMSCs were isolated and cultured by the whole bone marrow adherent method, followed by osteogenic and adipogenic induction and identification. The growth curve of passage 3 BMSCs was determined through the CCK-8 experiment. Passage 2 BMSCs were combined with Matrigel, and the morphology and proliferation of BMSCs were observed by hematoxylin-eosin staining under a phase contrast microscope. The viability of the cells was evaluated by the Live/Dead staining. RESULTS AND CONCLUSION:(1) BMSCs cultured by the whole bone marrow adherent method had the ability to differentiate into osteoblasts and adipocytes.(2) BMSCs exhibited an "S"-shaped growth curve, which was consistent with the growth characteristics of normal cells.(3) Under the phase contrast microscope, BMSCs cultured by the Matrigel model were extended and interconnected in a three-dimensional network growth state with good proliferation. The Matrigel gradually became soft and collapsed over time(7 days) and a few cells were still in a network growth after 14 days. Hematoxylin-eosin staining results showed that the cell cytoplasm was larger at 4 days and the cells became thinned and mutually cross-linked at 7 days.(4) The active percentage of BMSCs in the Matrigel model was 92.57%, 95.54% and 97.37% at 1, 4, 7 days of culture, respectively. To conclude, BMSCs cultured on the Matrigel has good proliferation and high viability.
引文
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[27]黄旋平,周诺,江献芳,等.人骨形态发生蛋白2基因修饰自体骨髓间充质干细胞移植促进兔下颌骨牵张成骨实验的组织形态学观察[J].中国组织工程研究,2012,16(23):4242-4246.
[2]Albanell J,Codony-Servat J,Rojo F,et al.Activated extracellular signal-regulated kinases:association with epidermal growth factor receptor/transforming growth factor alpha expression in head and neck squamous carcinoma and inhibition by anti-epidermal growth factor receptor treatments.Cancer Res.2001;61(17):6500-6510.
[3]廖锋,郑扬,耿彬,等.促进移植间充质干细胞成骨的研究进展[J].基础医学与临床,2013,33(10):1123-1126.
[4]Beyth S,Schroeder J,Liebergall M.Stem cells in bone diseases:current clinical practice.Br Med Bull.2011;99:199-210.
[5]Barnett MDJ,Pomeroy GC.Use of Platelet-Rich Plasma and Bone Marrow-Derived Mesenchymal Stem Cells in Foot and Ankle Surgery.Techniques in Foot&Ankle Surgery.2007;6(6):89-94.
[6]Kleinman HK,Martin GR.Matrigel:basement membrane matrix with biological activity.Semin Cancer Biol.2005;15(5):378-386.
[7]Mondrinos MJ,Koutzaki S,Jiwanmall E,et al.Engineering three-dimensional pulmonary tissue constructs.Tissue Eng.2006;12(4):717-728.
[8]张海峰,杜子婧,赵丹阳,等.绿色荧光蛋白基因转染示踪比格犬体内骨髓间充质干细胞分化[J].国际骨科学杂志,2016,37(2):117-122.
[9]Zhao J,Zhang Z,Wang S,et al.Apatite-coated silk fibroin scaffolds to healing mandibular border defects in canines.Bone.2009;45(3):517-527.
[10]Campagnoli C,Roberts IA,Kumar S,et al.Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood,liver,and bone marrow.Blood.2001;98(8):2396-2402.
[11]Lee OK,Kuo TK,Chen WM,et al.Isolation of multipotent mesenchymal stem cells from umbilical cord blood.Blood.2004;103(5):1669-1675.
[12]Jiang Y,Jahagirdar BN,Reinhardt RL,et al.Pluripotency of mesenchymal stem cells derived from adult marrow.Nature.2002;418(6893):41-49.
[13]No?l D,Gazit D,Bouquet C,et al.Short-term BMP-2expression is sufficient for in vivo osteochondral differentiation of mesenchymal stem cells.Stem Cells.2004;22(1):74-85.
[14]Silva GV,Litovsky S,Assad JA,et al.Mesenchymal stem cells differentiate into an endothelial phenotype,enhance vascular density,and improve heart function in a canine chronic ischemia model.Circulation.2005;111(2):150-156.
[15]Gafni Y,Turgeman G,Liebergal M,et al.Stem cells as vehicles for orthopedic gene therapy.Gene Ther.2004;11(4):417-426.
[16]Xu W,Zhang X,Qian H,et al.Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro.Exp Biol Med(Maywood).2004;229(7):623-631.
[17]Xu W,Qian H,Zhu W,et al.A novel tumor cell line cloned from mutated human embryonic bone marrow mesenchymal stem cells.Oncol Rep.2004;12(3):501-508.
[18]Neuhuber B,Swanger SA,Howard L,et al.Effects of plating density and culture time on bone marrow stromal cell characteristics.Exp Hematol.2008;36(9):1176-1185.
[19]Chen ZZ,Van Bockstaele DR,Buyssens N,et al.Stromal populations and fibrosis in human long-term bone marrow cultures.Leukemia.1991;5(9):772-781.
[20]李晓峰,赵劲民,苏伟,等.大鼠骨髓间充质干细胞的培养与鉴定[J].中国组织工程研究,2011,15(10):1721-1725.
[21]Aggarwal S,Pittenger MF.Human mesenchymal stem cells modulate allogeneic immune cell responses.Blood.2005;105(4):1815-1822.
[22]Brill E,Gobble R,Angeles C,et al.ZIC1 overexpression is oncogenic in liposarcoma.Cancer Res.2010;70(17):6891-6901.
[23]Kang BJ,Ryu HH,Park SS,et al.Effect of matrigel on the osteogenic potential of canine adipose tissue-derived mesenchymal stem cells.J Vet Med Sci.2012;74(7):827-836.
[24]Park SS,Lee YJ,Lee SH,et al.Functional recovery after spinal cord injury in dogs treated with a combination of Matrigel and neural-induced adipose-derived mesenchymal Stem cells.Cytotherapy.2012;14(5):584-597.
[25]Philp D,Chen SS,Fitzgerald W,et al.Complex extracellular matrices promote tissue-specific stem cell differentiation.Stem Cells.2005;23(2):288-296.
[26]Du WJ,Chi Y,Yang ZX,et al.Heterogeneity of proangiogenic features in mesenchymal stem cells derived from bone marrow,adipose tissue,umbilical cord,and placenta.Stem Cell Res Ther.2016;7(1):163.
[27]黄旋平,周诺,江献芳,等.人骨形态发生蛋白2基因修饰自体骨髓间充质干细胞移植促进兔下颌骨牵张成骨实验的组织形态学观察[J].中国组织工程研究,2012,16(23):4242-4246.