摘要
目的通过三种分离方法分离脊椎术中出血标本中的间充质干细胞并进行培养,比较得到的单核细胞(Mononuclearcell,MNC)数、细胞贴壁情况、原代培养时间、传代至第三代后得到的细胞总数,探索针对脊椎术中出血来源间充质干细胞理想的分离培养方法。为进一步实验及应用提供实验基础。方法以12例胸腰椎骨折病人为实验对象。分别取术中脊椎松质骨出血标本30ml随机分为3等份,标记为A、B、C三组。A组采用密度梯度法,B组采用甲基纤维素(Methylcellulose,MC)沉降法,C组采用密度梯度结合甲基纤维素的两步法分离MNC,用含10%胎牛血清的低糖DMEM培养基培养。72小时全量换液,以后3-5天按时换液,细胞达到汇合状态后进行胰蛋白酶消化传代。比较三种分离方法分离的MNC数、第三天贴壁细胞数、原代培养时间、第三代细胞数。倒置显微镜观察细胞形态及生长状态。流式细胞仪检测细胞表面标志鉴定细胞。结果用3种分离方法提取MNC数分别为:A组(6.99±0.77)×106,B组(11.5±1.70)×106,C组(8.20±0.96)×106,三组之间差异有统计学意义(P<0.05)。培养第三天观察细胞贴壁数分别为:A组11.40±2.37,B组为10.10±1.91,A组与B组两者之间的差异没有统计学意义(P>0.05);C组24±3.27,与其它两组比较有统计学差异(P<0.05)比较三组原代培养时间,A组为28.20±1.69天,B组为28.40±2.12天, A组与B组之间的差异没有统计学意义(P>0.05);C组为20.40±1.17天,与其它两组比较,差别有统计学意义(P<0.05)。传代培养3代后获得的细胞数,A组为(6.39±0.30)×106,B组为(5.54±0.42)×106, A组与B组比较没有统计学差异(P>0.05);C组为(8.43±0.30)×106,与其它两组比较,差别有统计学意义(P<0.05),三组培养细胞在倒置显微镜下观察,细胞形态一致,均为贴壁生长的长梭形或成纤维细胞样。流式细胞仪检测细胞表面标志,初次分离的细胞均有CD44表达阳性,CD34、CD45阳性率低,经过培养传代后均高表达CD29、CD44、CD90,而低表达CD14、CD34、CD45。结论脊椎术中松质骨出血标本中存在间充质干细胞,但数量较少,培养所得细胞经鉴定为间充质干细胞;甲基纤维素沉降结合密度梯度两步分离方法比其他两种方法有更好的培养效率,是分离单核细胞的较好方法;脊椎术中出血分离的间充质干细胞可以在体外进行有效的培养扩增,具有广泛的应用前景。
Objective This study is compare the effect with the different separation methods of mesenchymal stem cells (MSCs) in bleeding of thoracolumbar vertebral operation. The aim is to explore for the ideal method of MSCs separating from bleeding of spinal operation. Methods 12 patients whith thoracolumbar fracture were included in this experiment. The 30ml blood specimens from bleeding of cancellous bone in thoracolumbar vertebral operation of every patient were divided into three equal parts randomly and marked as A, B,C three groups respectively with different isolating methods:A group by density gradient seperation, B group with methyl cellulose (MC) precipitate seperation, C group by two-step seperation,which combined density gradient separation with MC precipitate seperation. The obtained MNC were cultured in low level sugar DMEM medium containing 10% fetal bovine serum. All medium was changed wholely after 72 hours and changed partly medium on time after 3-5 days. MNC were trypsined and passaged when these cells reached confluence state. MNC number of adherent cells on the third day, the primary culture time, the third generation of cells with three methods of separation wre compared. MNC morphology and their growth behavior were observed with inverted microscope. Cell surface markers of these cells were identified with flow cytometry. Results MNC number with three kinds of separation method were (6.99±0.77)×106 in A group,(11.5±1.70)×106 in B group and (8.20±0.96)×106 in C group. There is significant differences between the three groups (P<0.05). Adherent cells number on the third day were 11.40±2.37 in A group,10.10±1.91 in B group and 24±3.27 in C group. There was no significant differences between A group and B group (P>0.05), but there is significant differences between C group and anohter two groups (P<0.05).The primary culture time were 28.20±1.69 days in A group,28.40±2.12 days in B group and 20.40±1.17 days in C group. There is no significant differences between A group and B group (P>0.05), but there is significant differences between C group and anohter two groups (P<0.05). Subcultured third generations of cells were (6.39±0.30)×106 in A group, (5.54±0.42)×106 in B group and (8.43±0.30)×106 in C group. There is no significant differences between A group and B group (P>0.05), but there is significant differences between C group and anohter two groups (P<0.05). The cell morphology of three groups were the same feature:adherent longer spindle or fibroblastoid under inverted microscope. Cell surface markers were identified by flow cytometry. Cells were first isolated have CD44 expression always, however, positive rate of CD34, CD45 was lower. These MSCs after passage culture have highly CD29, CD44, CD90 expression, and lower expression of CD14, CD34, CD45. Conclusion There are cell identified as MSCs in the bleeding samples of thoracolumbar vertebral cancellous operation, but the number is very fewer. Two-step separation is a better separation of monocytes method. MSCs extract from bleeding in vertebral operation can be cultured in vitro amplification effectively and will have a wide range of application.
引文
[1]Pittenger MF, Mackay AM,Beck SC, et al. Multilineage potential of adult human mesenchymal stem cell. Science,1999,284(5411):143-147
[2]Hoglund M, Smedmyr B, Bengtsson, et al. Mobilization of CD34+cells by glycosylated and Nonglycosylated G-CSF in healthy volunteers:A compatative study. Eur J Haematol,1997,59:177-183
[3]Huss R, Lange C, Weissinger EM, et al. Evidence of peripheral blood derived, plastic adherent CD34, hematopoietic stem cell clones with mesenchymal stem cell characeristics[J]. Stem Cells,2000,18(4):252
[4]Zvaifler NJ, Marinova ML, Adams G, et al. Mesenchymal precursor cells in the blood of normal individuals[J]. Arthritis Res,2000,2(6):477
[5]Erices A,Conget P, Minguell JJ.Mesenchymal progenitor cells in human umbilical cord blood[J].Br J Haematol,2000,109(1):235
[6]Zuk PA, Zhu M, Mizuno H, et al.Multi-lineage cells from human adipose tissue:implications for cell based therapies. [J].Tissue Eng,2001,7(2):211
[7]Sefford KW, Hicok KC, Safford SD, et al.Neurongenic differentiation of marine and human adipose derived stromal cells[J].Biochem Biophys Res Commun,2002,294(2):371.
[8]Noort WA, Kruisselbrink AB, Anker PS, et al.Mesenchumal stem cells promote engraftment of human umbilical cord blood derived CD34+ cells in NOD/SCID mice [J].Exp Hematol,2002,30 (8):870-873
[9]Jiang Y, Vaessen B, Lenvi KT, et al. Multi-potent progentitor cells can be isolated from postnatal marine boen marrow, muscle and brain [J].Exp Hematol,2002,30(8):896-898
[10]Almeida PG, Elshabrawy D, Porada C, et al. Differentiative potential of human metanepric mesenchymal cells[J].Exp Hematol,2002,30(12):1454
[11]Wagner W, Wein F, Seckinger A, et al. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood.Exp Hematol 2005;33(11):1402-1416
[12]Romanov YA, Darevskaya AN, Merzlikina NV, et al.Mesenchymal stemcells from human bone marrow and adipose tissue:isolation,characterization,and differentiation potentialities.Bull Exp Biol Med2005;140(1):138-143
[13]MingueII JJ, Erices A, Conge TP. Mesenchymal stem cell.Exp Biolrichs Med 2001;226:507-520
[14]Yoo HJ, Yoon SS, Park S.Production and characterization of monoclonal antibodies to mesenchymal stem cells derived from human bone marrow.
Hybndoma(Larchmt)2005;24(2):92-97
[15]Pittenger MF, Mackay AM, Beck SC, et al.Multilineage potential of adult human mesenchymal stem cells.Cell Tissue Kinet,1987,20(3):263-272.
[16]Ouyang HW, Cao T, Zou XH, et al. Mesenchymal stem cell sheets revitalize nonviable dense grafts:implications for repair of large-bone and tendon defects. Transplantation.2006;82(2):170-174.
[17]Lopez-Heredia MA, Sohier J, Gaillard C, et al. Rapid prototyped porous titanium coated with calcium phosphate as a scaffold for bone tissue engineering. Biomaterials.2008;29(17):2608-2615.
[18]Noth U, Tuli BS, Osyczka. AM, et al. In vitro engineered cartilage constructs produced by press-coating biodegradable polymer with human
mesenchymal stem cells. Tissue Eng.2002;8(1):131-144.
[19]Zantop T, Gilbert TW, Yoder MC, et al. Extracellular matrix scaffolds
are repopulated by bone marrow-derived cells in a mouse model of achilles tendon reconstruction. J Orthop Res.2006;24(6):1299-1309.
[20]Schafer R, Wiskirchen J, Guo K, et al. Aptamer-based isolation and subsequent imaging of mesenchymal stem cells in ischemic myocard by magnetic resonance imaging. Rofo.2007;179(10):1009-1015.
[21]13. Rosenthal N.Youthful prospects for human stem-cell therapy. In
another few decades, revised attitudes toward stem cells could lead to disease prevention and life extension. EMBO Rep.2005;6:S30-34.
[22]Fadia A, Cassely LF, Sanchorawala V, et al. Incidence and outcome of acute renal failure complicating autologous stem cell transplantation for AL amyloidosis.Kidney Int 2003;63(5):1868-1873
[23]Masuya M, Drake CJ, Fleming PA, et al. Hematopoietic origin of glomerular mesangial cells. Blood.2003;101(6):2215-2218.
[24]Chen X, Lin X, Zhao J, et al. A tumor-selective biotherapy with Prol —onged impact on established metastases based on cytokine gene-engineered MSCs. Mol Ther.2008;16(4):749-756.
[25]Phinney DG. Isolation of mesenchymaI stem cells from murine bone marrow by immunodepletion. Methods MOI Biol,2008,449:171-186.
[26]Deryugin E I, Muller-Sieburg C E. Stromal cells in longterm cultures: kers to the elucidatopoietic development? [J]. Crit Rev Immunol,1993, 13:115-150.
[27]Kuznetsov SA, Mankani MH, Shi S, et al. Evidence for circulating osteogenic progenitors in humans and other mammals. Bone,1998,23(S5):211
[28]Zvaifler NJ, Marinova-Mutafchieva L, Adams G, et al. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res,2000,2(6):477-488.
[29]Reading L, Still K, Bishop N, et al. Peripheral blood as an alternative source of mesenchymal stem cells. Bone,2000,26(Suppl):9.
[30]Testa N, Molineux G. Clonal and long-term cultures using human bone marrow. In:Testa N, Molineux G, eds. Haemopoiesis. A Practical Approach. New York: Oxford University Press;1993. p.75-106.
[31]Soraya Carrancio, Natalia Lo'pez-Holgado,et al. Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification. Experimental Hematology 2008;36:1014-1021
[32]刘岱,赵玉明,晏晓青,崔雅宁,王晓军,徐军.人脐带血间充质干细胞分离培养体系的优化筛选[J].中国组织工程研究与临床康复,2009,3(10):1839-1843.
[33]田新,唐柳平,肖萍,符仁义,袁天柱,陈宏.脐血间充质干细胞分离扩增及向成骨与脂肪细胞的分化[J].中国组织工程研究与临床康复,中国组织工程研究与临床康复第2008 12(43)8593-8596.
[1]Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cell.Science,1999,284(5411):143-147 [2]Hoglund M, Smedmyr B, Bengtsson, et al. Mobilization of CD34+ cells by glycosylated and Nonglycosylated G-CSF in healthy volunteers:A compatative study. Eur J Haematol,1997,59:177-183
[3]Huss R, Lange C, Weissinger EM, et al. Evidence of peripheral blood derived, plastic adherent CD34,hematopoietic stem cell clones with mesenchymal stem cell characeristics[J].Stem Cells,2000,18(4):252
[4]Zvaifler NJ, Marinova ML, Adams G, et al. Mesenchymal precursor cells in the blood of normal individuals[J]. Arthritis Res,2000,2(6):477
[5]Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood[J].Br J Haematol,2000,109(1):235
[6]Zuk PA, Zhu M, Mizuno H, et al. Multi-lineage cells from human adipose tissue:implications for cell based therapies [J].Tissue Eng,2001,7(2):211
[7]Sefford KW, Hicok KC, Safford SD, et al. Neurongenic differentiation of marine and human adipose derived stromal cells[J].Biochem Biophys Res Commun,2002,294(2):371.
[8]Noort WA, Kruisselbrink AB, Anker PS,et al.Mesenchumal stem cells promote engraftment of human umbilical cord blood derived CD34+ cells in NOD/SCID mice [J].Exp Hematol,2002,30(8):870-873
[9]Jiang Y, Vaessen B, Lenvi KT, et al.Multi-potent progentitor cells can be isolated from postnatal marine boen marrow, muscle and brain [J].Exp Hematol,2002,30(8):896-898
[10]Almeida PG, Elshabrawy D, Porada C, et al.Differentiative potential of human metanepric mesenchymal cells[J].Exp Hematol,2002,30(12):1454
[11]Friedenstein AJ, Gorskaja JF, Kulagina NN.Fibroblast precurorsin normal and irradiated mouse hematopoietic organs.Exp Hematol,1976,4(5):267-274
[12]曾琴兵,程范军,陈龙,等.重组人促红细胞生成素促进体外培养的人骨髓间充质 干细胞的增殖.中华器官移植杂志.2009,30(1):21-25
[13]刘东宁,阴正勤,吴楠,等.Long-Evens大鼠骨髓间充质干细胞分离培养及生物学特性研究.第三军医大学学报,2008,30(15):1441-1443
[14]徐燕,慕晓玲.建立永生化骨髓间充质干细胞株.中国组织工程研究与临床康复,2007,11,(33):6552-6556.
[15]Pittenger MF, Mackay AM,Beck SC, et al.Multilineage potential of adult human mesenchymal stem cell.Science,1999,284(5411):143-147.
[16]Colter DC, Class R, Digirolama CM, et al.Rapid expansion of recycling stem cells in cultures of plastic adherent cells from human bone marrow.Proc Natl Acad Sci USA,2000,97(7):3123-3218.
[17]Gmnthos S, Zannettino AC, Hav SJ, et al.Molecular and ceIlular charaterisation of highly purified strmal stem cells derived from human bone marrow. Cell Sci 2003116(9):1827-1835.
[18]Muraglia A, Cancedda R,Quano R.Clonal mesenchymal progenitors from human bone marrow defferentiate in vitro according to ahierarchical model.Cell Sci,2000,113(7):1161-1166.
[19]Barda-Saad M, Shav-Tal Y. Rozenszain AL,et al.The mesenchymal expresses T cell receptor mRNA:relevance to cell growth control.Oncogen 2002, 21(13):2029-2036.
[20]Campagnoli C,Roberts IA., Kumar S, et al. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood,livel and bone marrow.Blood 2001,98(8):2396-2402.
[21]Baksh D. Davies JE. Zandstra PW. Adult human bone marrow-derived mesenchymal progenitor cells are capable of adhesion-independen survival and expansion. Exp Hematol,2003,31:723-732.
[22]Deans RJ, Moseley AB. Mesenchymal stem cells:biology and potential clinical uses[J]. Exp Hematol,2000,28(8):875-884.
[23]Haynesworth SE, Baber MA, Caplan AI, et al. Spatial and temporal distribution of CD44 and osteopontin in fracture callus [J].J Cell Physiol,1996,166(3):585-592.
[24]Yamazaki M, Nakajima F, Ogasawara A, et al.Spatial and temporal distribution of CD44 and osteopontin in fracture callus [J].J Bone Joint Surg Br,1999,81(3):508-515.
[25]Deans RJ,Moseley AB. Mesenchymal stem cells:biology and potential clinical uses. Exp Hematol,2000,28(8):875-884.
[26]Pittenger MF, Mackay AM, Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Cell Tissue Kinet,1987,20(3):263-272.
[27]Phinney DG. Isolation of mesenchymaI stem cells from murine bone marrow by immunodepletion.Methods MOI Biol,2008,449:171-186.
[28]Lennon D P. A chemically defined medium supports in vitro proliferation and maintains the osteochondral potential of rat marroiw-derived mesenchymal stem cells[J].Exp Cell Res,1995,219:211-222.
[29]Meuleman N, Tondreau T, Delforge A,et al. Human marrow mesenchymal stem cell culture:serum-free medium allows better expansion than classical a-MEM medium. Eur J Haematol 2006,76:309-316.
[30]Katharina Schallmoser, Christina Bartmann, Eva Rohde, et al. Human platelet lysate can replace fetal bovine serum for clinical-scale Expansion of functional mesenchymal strmal cells. Transfusion,2007, 47:1436-1446
[31]Ouyang HW,Cao T, Zou XH, et al. Mesenchymal stem cell sheets revitalize nonviable dense grafts:implications for large bone and tendon defects.Transplantation,2006,82(2):170-174.
[32]Lopez-Heredia MA, Sohier J, Galard C, et al.Rapid prototyped porous titanium caoted with calciun phosphate as a scaffold for boen tissue engineering.Biomaterials,2008,9(17):2608-2615.
[33]Awad HA,Butler DL,Hams MT et a.In vitro characterization of mesenchymal stem cell-seeded collagen scaffolds for tendon repair:effects of initial seeding density on contraction kinetics.Biomed Mater Res,2000,51(2): 233-240.
[34]zantop T,Gilbert TW,Yoder MC,et al.Extracellular matrix scaffolds are repopulated by bone marrow-derived cells in a mouse model of achilles tendon reconstruction.Orthop Res,2006,24(6):1299-1309.
[35]Mohyeddin-Bonab M,Mohamad-Hassani MR,Alimoghaddam K.Autologous in vitro expanded mesenchymal stem cell therapy for human old mocardial infarction [J]. Arch Iran Med,2007,10(4):467-473
[36]Hirsch A,Nijveldt R,Vander Vleuten PA,et al.Inteacoronary infusion of autologous mononuclear bone marrow cells in patients with acute myocafdial infarction treated with primary PCI:Pilot study of the multicenter HEBE trial.catheter Cardiovasc Interv,2008,71(3): 273-281
[37]Mezey E,Chamdross KJ.Harta G,et al.Turning blood into brain:cells bearing neuromal antigens generated in viro from bone marrow.Science,2006,290(5497):1779-1782
[38]Aziz SR,ziccardi VB,Borah G,et al.Current therapy:complications associated with rigid intemal fixation of facial fractures.Compend Contin Educ Dent,2005,26(8):565-571.
[39]陈秉耀,赵旭红,侯树勋,等.大鼠损伤脊髓内异体骨髓间充质干细胞移植后的存活、迁移及分化。中华创伤骨科杂志,2008,10(10):951-954.
[40]Yoon SH, Shim YS, Park YH, et al. Complete spinal cord injury treatment using autologou bone marrow cell transplantation and bone marrow simulation with granulocyte-macrophage colony stimulating factor:Phase Ⅰ/Ⅱclinical trial.Stem Cells,2007,25:2066-2073
[41]Lee K, Majumdar MK, Buyaner D, et al. Human mesenchymal stem cells maintain transgene expression during expanstion and differentiation.Mol Ther, 2001,3(6):857-866.
[42]Liebemlan JR, Le LQ. Wu L, et al. Regional gene therapy with a BMP-2-producing murine stromal cell line induces heterotopic and orthotopic bone fomation in rodents. J Orthop Res,1998,16(3):330-339,
[43]Chen X, Lin X, Zhao J, et al.A tumor-selective biotherapy with prolonged impact on established metastases based on cytokine gene-engineered MSCs. Mol Ther,2008,16(4):749-756