摘要
背景:研究表明,低浓度四环素可促进体外培养的人胚成骨细胞活性及增殖,增加且加强细胞合成Ⅰ型胶原、骨钙素等促进骨生成的物质。目的:观察盐酸四环素体外对大鼠骨髓间充质干细胞增殖的作用。方法:取第3代SD大鼠骨髓间充质干细胞,分2组干预,对照组加入含胎牛血清的完全培养基培养,实验组加入含盐酸四环素及胎牛血清的完全培养基培养。培养1-8 d,采用MTT法、细胞计数法检测两组细胞增殖活性。结果与结论:(1)细胞计数法:随着培养时间的延长,两组细胞数量逐渐增加,实验组培养4-8 d的细胞数量高于对照组(P<0.05);(2)MTT法:两组细胞数量均随培养时间的延长而增加,第1天为细胞潜伏期,3-6 d为对数生长期,第7天,对照组细胞进入平台期,实验组细胞继续增殖,实验组培养5-8 d的细胞增殖高于对照组(P<0.05);(3)结果表明:盐酸四环素可促进骨髓间充质干细胞的增殖。
BACKGROUND: Tetracycline hydrochloride is shown to promote viability and proliferation of human embryonic osteoblasts cultured in vitro, increase the production of type I collage and osteocalcin. OBJECTIVE: To study the proliferation activity of bone marrow mesenchymal stem cells(BMSCs) cultured by tetracycline hydrochloride in vitro. METHODS: Passage 3 BMSCs from Sprague-Dawley rats were cultured and divided into two groups: cells in control group were cultured in complete medium containing fetal bovine serum, and those in experimental group were cultured in complete medium containing tetracycline hydrochloride. After days 1-8 of culture, MTT and cell counting assay were used to detect cell viability and proliferation in the two groups. RESULTS AND CONCLUSION:(1) Cell counting assay: with time, the number of cells in both groups was increased, but the cell number in the experimental group was higher than that in the control group at days 4-8 of culture(P < 0.05).(2) MTT assay: With time, the number of cells in both groups was increased: incubation period was at day 1, logarithmic growth period was within days 3-6, and plateau period began at day 7. Then, the cells continued to proliferate, and the cell proliferation was higher in the experimental group than the control group at days 5-8 of culture(P < 0.05). To conclude, tetracycline hydrochloride can significantly enhance BMSCs proliferation.
引文
[1]Golub LM,Lee HM,Lehrer G,et al.Minocycline reduces gingival collagenolytic activity during diabetes:preliminary observations and a proposed new mechanism of action.J Periodontal Res.1983;18(5):516-526.
[2]Golub LM,Ramamurthy NS,Mc Namara TF,et al.Tetracyclines inhibit tissue collagenase activity:a new mechanism in the treatment of periodontal disease.J Periodontal Res.1984;19:651-655.
[3]Golub LM,Goodson JM,Lee HM,et al.Locally and lowdose systemically administered tetracycline inhibitt issue collagenase activity:potential new approaches in the treatment of periodontal disease.J Periodontol.1985;56:93-97.
[4]Golub LM,Wolf M,Lee HM,et al.Further evidence that Tetracyclines inhibit collagenase activity in human crevicular fluid and from other mammalian source.J Periodont Res.1985;20:12-23.
[5]Kaneko H,Sasaki T,Ramamurthy NS,et al.Tetracycline administration normalizes the struction and acid phosphatase activity of osteoclasts in sterptozotocin-induced diabetes rats.Antomical Record.1990,227:427.
[6]Gerson SL.Mesenchymal stem cells:no longer second class marrow citizens.Nat Med.1999;5(3):262-264.
[7]Bonab MM,Alimoghaddam K,Talebian F,et al.Aging of mesenchymal stem cell in vitro.BMC Cell Biol.2006;7:14.
[8]孔清泉,项舟,杨志明.骨髓基质干细胞作软组织工程种子细胞研究[J].中国修复重建外科杂志,2002,16(4):277-280.
[9]陈旭,杨志明,解慧琪,等.WO-1对成骨细胞的生物学效应研究[J].中国修复重建外科杂志,2005,19(10):822-825
[10]李莉,黄永灿,常丽,等.盐酸四环素缓释微球的制备[J].华西药学杂志,2008,23(4):391-393.
[11]张珏,陈晓和,李莉,等.盐酸四环素缓释微球对大鼠成骨细胞活性的影响[J].华西药学杂志,2010,25(1):1-3.
[12]张珏,邓媛媛,曾富佳.盐酸四环素缓释微球对成骨细胞增殖作用的研究[J].中国民族民间医药杂志,2013,22(18):19-20.
[13]李宁,李应福,谢兴文,等.中药诱导骨髓间充质干细胞的定向分化[J].中国组织工程研究,2016,20(1):135-139.
[14]鲍远,黄俊明,靖兴志,等.淫羊藿苷促进骨髓间充质干细胞成骨分化[J].中国组织工程研究,2016,20(24):3501-3507.
[15]Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science.1999;284(5411):143-147.
[16]Prockop DJ.Marrow stromal cells as stem cells for nonhematopoietic tissues.Science.1997;276(5309):71-74.
[17]Richards M,Huibregtse BA,Caplan AI,et al.Marrow-derived progenitor cell injections enhance new bone formation during distraction.J Orthop Res.1999;17(6):900-908.
[18]Fukuda K.Molecular characterization of regenerated cardiomyocytes derived from adult mesenchymal stem cells.Congenit Anom(Kyoto).2002;42(1):1-9.
[19]Lu P,Blesch A,Tuszynski MH.Induction of bone marrow stromal cells to neurons:differentiation,transdifferentiation,or artifact.J Neurosci Res.2004;77(2):174-191.
[20]Clabaut A,Delplace S,Chauveau C,et al.Human osteoblasts derived from mesenchymal stem cells express adipogenic markers upon coculture with bone marrow adipocytes.Differentiation.2010;80(1):40-45.
[21]Cipriani P,Guiducci S,Miniati I,et al.Impairment of endothelial cell differentiation from bone marrow-derived mesenchymal stem cells:new insight into the pathogenesis of systemic sclerosis.Arthritis Rheum.2007;56(6):1994-2004.
[22]Ouyang JF,Lou J,Yan C,et al.In-vitro promoted differentiation of mesenchymal stem cells towards hepatocytes induced by salidroside.J Pharm Pharmacol.2010;62(4):530-538.
[23]杨志明.组织工程基础与临床[M].成都:四川科学技术出版社,2000:4.
[24]Xu S,De Becker A,Van Camp B,et al.An improved harvest and in vitro expansion protocol for murine bone marrow-derived mesenchymal stem cells.J Biomed Biotechnol.2010;2010:105940.
[25]Chi LLY,Bianco J,Giardini-Rosa R,et al.Direct and indirect co-culture of bone marrow stem cells and adipose-derived stem cells with chondrocytes in 3D scaffold-free culture.J Regen Med Tissue Eng.2016;2050-1218(5):1-5.
[26]Zhao Y,Waldman SD,Flynn LE.Multilineage co-culture of adiposederived stem cells for tissue engineering.J Tissue Eng Regen Med.2015;9:826-837.
[27]Boyle J,Luan B,Cruz TF,et al.Characterization of proteoglycan accumulation during formation of cartilagenous tissue in vitro.Osteoarthritis Cartilage.1995;3:117-125.
[28]张岩,陈曦海,纪艳超,等.贴壁法体外分离培养大鼠间充质干细胞的效果验证[J].中国组织工程研究与临床康复,2010,14(6):1005-1008.
[29]Zuk PA,Zhu M,Mizuno H,et al.Multilineage cells from human adipose tissue:implications for cell-based therapies.Tissue Eng.2001;7:211-228.
[30]Flynn L,Prestwich GD,Semple JL,et al.Adipose tissue engineering with naturally derived scaffolds and adipose-derived stem cells.Biomaterials.2007;28:3834-3842.
[31]石淙,万腊根.细胞增殖的检测方法[J].实验与检验医学,2012,30(2):153-155.
[32]Goel H0C,Prakash H,Ali A,et al.Podophyllum hexandrum modu-lates gamma radiation-induced immunosuppression in Balb/c mice:implications in radioprotection.Mol Cell Biochem.2007;295(1-2):93-103.
[33]Lin P,Allison DC.Measurement of DNA content and of tritiated thymidine and bromodeoxyuridine incorporation by the same cells.J Ilistochem Cytochem.1993;41(9):1435-1439.
[34]赵嘉惠,张华屏,王春芳.MTT法在检测细胞增殖方面的探讨[J].山西医科大学学报,2007,38(3):262-264.
[35]文珠,胡国柱,俞火,等.黄精多糖干预长春新碱抑制骨髓基质细胞增殖的研究[J].中华中医药杂志,2011,26(7):1630-1632.
[36]Westendorf JJ,Kahler RA,Schroeder TM.Wnt signaling in osteoblasts and bone diseases.Gene.2004;341:19-39.
[37]侯费祎,谢兴文,席芳琴.等.麝香酮含药血清对大鼠骨髓间充质干细胞的增殖、分化的影响[J].西安交通大学学报,2013,34(1):110-114.
[38]Burrow KL,Hoyland JA,Richardson SM.Human Adipose-Derived Stem Cells Exhibit Enhanced Proliferative Capacity and Retain Multipotency Longer than Donor-Matched Bone Marrow Mesenchymal Stem Cells during Expansion In Vitro.Stem Cells Int.2017;2017:2541275.
[39]Sherman AB,Gilger BC,Berglund AK,et al.Effect of bone marrow-derived mesenchymal stem cells and stem cell supernatant on equine corneal wound healing in vitro.Stem Cell Res Ther.2017;8(1):120.
[40]da Silveira Gerzson A,Machado DC,Marinovic DR,et al Assessment of Adhesion and Proliferation of Bone Marrow Mesenchymal Stem Cells in Polymer Matrices with rh GH.Int J Oral Maxillofac Implants.2017;32(3):e183-e189.
[41]Danisovic L,Oravcova L,Krajciova L,et al.Effect of long-term culture on the biological and morphological characteristics of human adipose tissue-derived stem Cells.J Physiol Pharmacol.2017;68(1):149-158.
[42]Blázquez-Prunera A,Díez JM,Gajardo R,et al.Human mesenchymal stem cells maintain their phenotype,multipotentiality,and genetic stability when cultured using a defined xeno-free human plasma fraction.Stem Cell Res Ther.2017;8(1):103.
[43]Batsali AK,Pontikoglou C,Koutroulakis D,et al.Differential expression of cell cycle and WNT pathway-related genes accounts for differences in the growth and differentiation potential of Wharton's jelly and bone marrow-derived mesenchymal stem cells.Stem Cell Res Ther.2017;8(1):102.
[44]Maadawi ZME.Conditioned Medium Derived from Salidroside-Pretreated Mesenchymal Stem Cell Culture Ameliorates Mouse Lipopolysaccharide-Induced Cerebral Neuroinflammation-Histological and Immunohistochemical Study.Int J Stem Cells.2017;10(1):60-68.
[45]Zeng YL,Zheng H,Chen QR,et al.Bone marrow-derived mesenchymal stem cells overexpressing Mi R-21 efficiently repair myocardial damage in rats.Oncotarget.2017;8(17):29161-29173.
[46]Futrega K,Atkinson K,Lott WB,et al.Spheroid Coculture of Hematopoietic Stem/Progenitor Cells and Monolayer Expanded Mesenchymal Stem/Stromal Cells in Polydimethylsiloxane Microwells Modestly Improves In Vitro Hematopoietic Stem/Progenitor Cell Expansion.Tissue Eng Part C Methods.2017;23(4):200-218.
[47]Bottagisio M,Lopa S,Granata V,et al.Different combinations of growth factors for the tenogenic differentiation of bone marrow mesenchymal stem cells in monolayer culture and in fibrin-based three-dimensional constructs.Differentiation.2017;95:44-53.
[48]Cleary MA,Narcisi R,Albiero A,et al.Dynamic Regulation of TWIST1 Expression During Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells.Stem Cells Dev.2017;26(10):751-761.
[49]Shou K,Niu Y,Zheng X,et al.Enhancement of Bone-Marrow-Derived Mesenchymal Stem Cell Angiogenic Capacity by NPWT for a Combinatorial Therapy to Promote Wound Healing with Large Defect.Biomed Res Int.2017;2017:7920265.
[50]Perucca S,Di Palma A,Piccaluga PP,et al.Mesenchymal stromal cells(MSCs)induce ex vivo proliferation and erythroid commitment of cord blood haematopoietic stem cells(CB-CD34+cells).PLo S One.2017;12(2):e0172430.
[51]Wang MY,Nestvold J,Rekdal?,et al.A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties.Exp Cell Res.2017;352(2):218-224.
[52]Tsai TL,Li WJ.Identification of Bone Marrow-Derived Soluble Factors Regulating Human Mesenchymal Stem Cells for Bone Regeneration.Stem Cell Reports.2017;8(2):387-400.