玻璃粘连蛋白对大鼠骨髓间充质干细胞成骨分化的影响
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摘要
目的：体外分离纯化培养大鼠骨髓间充质干细胞(rMSC),并进一步研究玻璃粘连蛋白(vitronectin VN)在干细胞向成骨方向分化过程中的作用。方法：贴壁分离培养法分离纯化rMSC,再用以玻璃粘连蛋白包被的培养板培养,定期检测碱性磷酸酶活性,钙结节的形成。结果：经贴壁筛选法分离的rMSC生长曲线呈S型,第3、4、5代细胞排列具有典型的漩涡状结构,表达CD44、CD90,不表达CD34,电镜观察为低分化细胞,用以玻璃粘连蛋白包被的培养板培养12天,碱性磷酸酶染色呈阳性,硝酸银染色显示有钙结节形成。结论：玻璃粘连蛋白有诱导rMSC向成骨方向分化的能力。
Objective:TO isolate, purify and amplify of rat mesenehymal stem cells (rMSCs)in vitro, and further studying the effect of vitronectin (VN)on osteogenic diferentiation of rat mesenchymalstem cells. Methods:RMSCs were isolated and purified by wall adhesion culture procedure and passaged in vitro. The cells of the third passage were cultured in cultureplates coated by vitronectin, and detecting calcifying nodule and alkaline phosphatase (ALP) activity regularly. Rsults:The rMSCs grows the curve reports the S form, and 3rd,4th and 5th generation were very purified and all in the whirlpool-shape through isolation, purification and araplitieation. These cultured cells showed immunoreactivity to CD44 and CD90 but not CD34 and low-differentiation under the electron microscopy. The ALP expression and calcifying nodule were strongly positive of there cells cultured 12 days in cultureplates coated by vitronectin. Conclusion:Vitronectin has the ability of inducing osteogenic diferentiation of rat mesenchymalstem cells.
Objective:TO isolate, purify and amplify of rat mesenehymal stem cells (rMSCs)in vitro, and further studying the effect of vitronectin (VN)on osteogenic diferentiation of rat mesenchymalstem cells. Methods:RMSCs were isolated and purified by wall adhesion culture procedure and passaged in vitro. The cells of the third passage were cultured in cultureplates coated by vitronectin, and detecting calcifying nodule and alkaline phosphatase (ALP) activity regularly. Rsults:The rMSCs grows the curve reports the S form, and 3rd,4th and 5th generation were very purified and all in the whirlpool-shape through isolation, purification and araplitieation. These cultured cells showed immunoreactivity to CD44 and CD90 but not CD34 and low-differentiation under the electron microscopy. The ALP expression and calcifying nodule were strongly positive of there cells cultured 12 days in cultureplates coated by vitronectin. Conclusion:Vitronectin has the ability of inducing osteogenic diferentiation of rat mesenchymalstem cells.
引文
[1]Pittenger MF, Packay AM, Beck SC, et al Mutilinage potential of adult human mesenchymal stem cell (J) Science,1999; 284(5411):143 7
[2]Rockop DJ Marrow stromal cells as stem cells form nonhematopoietic tissues (J) Science,1997; 276(5309):71 4
[3]Bruder S.P.Jaiswal N,HaynesworthS.E.Growth kinetics,self-renewal,and the osteogenic potential of purified human mesenchymal stem cells during extensive sub cultivation and following cryopreservation. J Cell Biochem.1997;64(2):278-294.
[4]Bortell R, Barone L.M, Tassinari M.S, Lian J.B, Stein G.S. Gene expression during endochondral bone development:evidence for coordinate expression of transforming growth factor beta 1 and collagen type I. J Cell Biochem.1990;44(2):81-91.
[5]Mizuno M, Kuboki Y. Osteoblast-related gene expression of bone marrow cells during the osteoblastic differentiation induced by type I collagen. J Biochem (Tokyo). 2001;129(1):133-138.
[6]CATELAS I, SESE N, WU B M, et al. Human mesenchymal stem cell proliferation and osteogenic differentiation in fibrin gels in vitro [J]. Tissue Eng,2006,12 (8):2385-2396.
[7]刘斌钰,李宁毅,樊功为,等.大鼠骨髓间充质干细胞的培养鉴定及向成骨细胞诱导分化研究[J].齐鲁医学杂志,2007,22(3)：215-217.
[8]Hong JH, Yaffe MB. TAZ:a beta-catenin-like molecule that regulates mesenchymal stem cell differentiation. Cell cycle (Georgetown, Tex.),2006,5(2):176-9.
[9]叶发刚,夏长所,张卫兵,等.兔骨髓间充质干细胞的分离、培养与鉴定[J].青岛大学医学院学报,2006,42(4)：330-332.
1、Thomson JA,ItskovitzEldor J.Shapiro SS et al.Embryonic ste m cell lines derived from human blastocysts [J]. Science,1998,282:1145～114.
2、Filip S,Morkr J,Hrusk A,Adult Stem Cells and Their Importance in Cell Therap y [J].Folia Biologica,2003,49:9-14.
3、Ingrid K,Margaret AG.The therapeutic potential of stem cells from adults.BMJ.2002,325:372～376 Elaine F,Julia AS.Stem Cells:A New Lease on Life [J]. Cell 2000,100:143～155.
4、Andrades J A, Han B.A recombinant human TGF betel fusion protein with collagen binding domain promotes migration, growth, and differentiation of bone marrow mesenchymal cells\[J\].Exp Cell Res,1999,250(2):485-498.
5、Mason J M, Breitbart A S, Barcia M, et al. Cartilage and bone regeneration using gene-enhanced tissue engineering\[J\]. Clin Orthop,2000,379:171-178.
6、Gao J, Dennis J E, Solchaga LA, et al. Tissue engineered fabrication of an osteochondral composite graft using rat bone marrow derived mesenchymal stem cells\[J\].Tissue Eng,2001,7(4):363-371.
7、戚超,杨志明,黄富国,等.骨髓基质干细胞与生物衍生骨复合修复山羊股骨缺损的研究＼[J＼].中国修复重建外科杂志,2005,19：90-94.
8、Phinney DG, Kopen G, Isaacson RI,et al. Plastic adherent stromal cells from the bone marrow of commonly used straing of inbred mice:variations in yield, growth and differentiation [J]. J Cell Biochem,1999,72:570.
9、Aubin JF. Steoprogenitor cell frequency in rat bone marrow stromal populations, role for heterotypic cell cell interaction in osteoblast differentiation [J]. Cell Tissue Res, 1999,73(3):396 401.
10、Otsuka E, Yamaguchi A, Shigehisa H, et al. Characterization of osteoblastic differentiation of stromal cell line ST2 that is induced by ascorbic acid [J]. Am J Physiol, 1999,277(Cell Physiol 46):C132.
11、McQilla DJ, Richardson MD, Bateman JF. Matrix deposition by a calcifying human osteogenic sarcoma cell line(SAGS 2) [J]. Bone,1995,16:415.
12、Maniatopoulos C, Sodek J, Melcher AH, et al. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats [J]. Tissue Res,1998,254(2):317.
13、Fried Benayahu D. Dexamethasone regulation of marrow stromal-derived osteoblast cells [J]. J Cell Biochem,1996,62:476.
14、Alborzi A, Mac K, Glackin CA,et al. Endochondral and intramembranous fetal bone development:osteoblastic cell proliferation and expression of alkaline phosphatase, mtwist, and histone H4 [J]. J Craniofac Gent Dev Biol,1996,6(2):94.
15、Al GP, SHU YP, YANG GH,et al. Osteogenic and chondrogenic differentiation of mesenchymal cells from swine bone marrow [J]. Zhongguo Linchuang Kangfu(Chin J Clin Rehabil),2002,6(18):2689.
16、JIANG Y, JAHAGIRDAR B N, REINHARDT R L, et al. Pluripotency of mesenchymal stem cells derived from adult marrow [J]. Nature,2002,418:41-49
17、王洪复.骨细胞图谱与骨细胞体外培养技术[M].上海：上海科学技术出版社,2001：60-64.
18、ZHANG B, WANG F, DENG L, et al. Isolating and culturing rat Marrow mesenchymal stem cells and studying their phenotypical and functional properties [J]. Journal of West China University of Medical Sciences,2003,34 (4):738
19、LISIGNOSI G, REMIDDI G, CATTINI L, et al. Anelevatednumber of differentiated osteoblast colonies can be obtained from rat bone marrow stromal cells using a gradient isolation procedure [J]. Connect Tissue Res,2001,42 (1):49.
20、FRIEDENSTEIN A J, CHAILAKHYAN R K, GERASIMOV U V. Bone marrow osteogenic stem cells:in vitro cultivation and transplantation in diffusion chambers [J] Cell Tissue Kinet,1987,20 (3):26-27.
21、刘斌钰,李宁毅,樊功为,等.大鼠骨髓间充质干细胞的培养鉴定及向成骨细胞诱导分化研究[J].齐鲁医学杂志,2007,22(3)：215-217.
22、DERYUGINA E L, MULLER-SIEBURG C E. Stromal cells in long term cultures: keys to the elucidation of hematopoietic development [J]. Crit Rev Immunol, 1993,13:115-120.
23、PEISTER A, MELLAD J A, LARSON B L. Adult stem cells from bone marrow (MSCS) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential [J]. Blood,2004,103 (5):1662-1668.
24、GUO Z, YANG J, LIU X, et al. Biological features of mesenchymal stem cells from human bone marrow [J].Chin Med J (Engl),2001,114 (9):950-953
25、MANIATOPOULOS C, SODEK J, MELCHER A H. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats [J]. Cell Tissue Res, 1988,254 (2):314-317.
26、陈光兴,杨柳,周红星,等.间充质细胞与骨组织共培养条件下的成骨特性[J].中国临床康复,2004,8(8)：1433-1435
27、阮绪芝,陈霞萍,严世荣,等.骨髓基质干细胞的分离纯化及培养[J].生物学杂志,2004,21(2)：25-27.
28、KNIPPENBERG M, HELDER M N, DOULABI B Z, et al. Adipose tissue-derived mesenchymal stem cells acquire bone cell-like responsiveness to fluid shear stress on osteogenic stim-ulation[J]. Tissue Engin,2005,11 (11-12):1780-1788.
29、LIN H T, TARNG Y W, CHEN Y C, et al. Using human plasma supplemented medium to cultivate human bone mar-row-derived mesenchymal stem cell and evaluation of its multi-ple-lineage potential [J]. Transplant Proc,2005,37 (10):4504-4505.
30、 张文,田杰,江德勤,等.骨髓MSC体外分化为心肌样细胞相关调控基因的时序表达[J].中华心血管病杂志,2004,32(11)：1004-1008.
31、KITANO Y,'RADU A, SHABBAN A, et al. Selection, en-richment and culture expansion of murine mesenchymal pro-genitor cells by retroviral transduction of cycline adherent bone marrow cells [J]. Exp Hematol,2000,28 (12):1460-1469
32、GUO X M, WANG C Y, WANG Y H, et al. Experimental study of the isolation, culture and in chondrogenic differentia-tion of human bone mesenchymal stem cell [J] Chin J Stoma-tol,2003,38 (1):63-66.
33、WULF G G, JACKSON K A, GOODELL M A. Somatic stem cell plasticity: current evidence and emerging concepts [J]. Exp Hematol,2001,29 (12):1361-1370.
34、BARRY F P. Mesenchynal stem cell therapy in joint disease [J]. Novartis Found Symp,2003,249:86-96.
35、Pittenger MF, Packay AM, Beck SC, et al Mutilinage potential of adult human mesenchymal stem cell (J) Science,1999; 284(5411):143 7
36、Rockop DJ Marrow stromal cells as stem cells form nonhematopoietic tissues (J) Science,1997; 276(5309)71 4
37、Kopen GC, Prockop DJ, Phinney DG Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains (J) Proc Natl Acad Sci USA,1999; 96(19):10711 6
38、Brazelton TR, Rossi FMV, Keshet GI, et al From marrow to brain:expression of neuronal phenotypes in adult mice (J) Science,2000; 290(5497):1775 9
39、Ankeny DP,McTigue DM, Jakeman LB,et al Bone marrow transplants provide tissue protection and directional guidance for axons after contusive spinal cord injury in rats (J) Exp Neurol,2004; 190(1):17 31
40、Hofstetter CP, Schwarz EJ, Hess D, et al Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery (J) PNAS,2002; 99(4):2199 204
41、丁亮华,罗光华,董选.成人骨髓基质干细胞体外诱导成骨细胞＼[J＼].江苏医药,2005,2：84-86.
[2]Rockop DJ Marrow stromal cells as stem cells form nonhematopoietic tissues (J) Science,1997; 276(5309):71 4
[3]Bruder S.P.Jaiswal N,HaynesworthS.E.Growth kinetics,self-renewal,and the osteogenic potential of purified human mesenchymal stem cells during extensive sub cultivation and following cryopreservation. J Cell Biochem.1997;64(2):278-294.
[4]Bortell R, Barone L.M, Tassinari M.S, Lian J.B, Stein G.S. Gene expression during endochondral bone development:evidence for coordinate expression of transforming growth factor beta 1 and collagen type I. J Cell Biochem.1990;44(2):81-91.
[5]Mizuno M, Kuboki Y. Osteoblast-related gene expression of bone marrow cells during the osteoblastic differentiation induced by type I collagen. J Biochem (Tokyo). 2001;129(1):133-138.
[6]CATELAS I, SESE N, WU B M, et al. Human mesenchymal stem cell proliferation and osteogenic differentiation in fibrin gels in vitro [J]. Tissue Eng,2006,12 (8):2385-2396.
[7]刘斌钰,李宁毅,樊功为,等.大鼠骨髓间充质干细胞的培养鉴定及向成骨细胞诱导分化研究[J].齐鲁医学杂志,2007,22(3)：215-217.
[8]Hong JH, Yaffe MB. TAZ:a beta-catenin-like molecule that regulates mesenchymal stem cell differentiation. Cell cycle (Georgetown, Tex.),2006,5(2):176-9.
[9]叶发刚,夏长所,张卫兵,等.兔骨髓间充质干细胞的分离、培养与鉴定[J].青岛大学医学院学报,2006,42(4)：330-332.
1、Thomson JA,ItskovitzEldor J.Shapiro SS et al.Embryonic ste m cell lines derived from human blastocysts [J]. Science,1998,282:1145～114.
2、Filip S,Morkr J,Hrusk A,Adult Stem Cells and Their Importance in Cell Therap y [J].Folia Biologica,2003,49:9-14.
3、Ingrid K,Margaret AG.The therapeutic potential of stem cells from adults.BMJ.2002,325:372～376 Elaine F,Julia AS.Stem Cells:A New Lease on Life [J]. Cell 2000,100:143～155.
4、Andrades J A, Han B.A recombinant human TGF betel fusion protein with collagen binding domain promotes migration, growth, and differentiation of bone marrow mesenchymal cells\[J\].Exp Cell Res,1999,250(2):485-498.
5、Mason J M, Breitbart A S, Barcia M, et al. Cartilage and bone regeneration using gene-enhanced tissue engineering\[J\]. Clin Orthop,2000,379:171-178.
6、Gao J, Dennis J E, Solchaga LA, et al. Tissue engineered fabrication of an osteochondral composite graft using rat bone marrow derived mesenchymal stem cells\[J\].Tissue Eng,2001,7(4):363-371.
7、戚超,杨志明,黄富国,等.骨髓基质干细胞与生物衍生骨复合修复山羊股骨缺损的研究＼[J＼].中国修复重建外科杂志,2005,19：90-94.
8、Phinney DG, Kopen G, Isaacson RI,et al. Plastic adherent stromal cells from the bone marrow of commonly used straing of inbred mice:variations in yield, growth and differentiation [J]. J Cell Biochem,1999,72:570.
9、Aubin JF. Steoprogenitor cell frequency in rat bone marrow stromal populations, role for heterotypic cell cell interaction in osteoblast differentiation [J]. Cell Tissue Res, 1999,73(3):396 401.
10、Otsuka E, Yamaguchi A, Shigehisa H, et al. Characterization of osteoblastic differentiation of stromal cell line ST2 that is induced by ascorbic acid [J]. Am J Physiol, 1999,277(Cell Physiol 46):C132.
11、McQilla DJ, Richardson MD, Bateman JF. Matrix deposition by a calcifying human osteogenic sarcoma cell line(SAGS 2) [J]. Bone,1995,16:415.
12、Maniatopoulos C, Sodek J, Melcher AH, et al. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats [J]. Tissue Res,1998,254(2):317.
13、Fried Benayahu D. Dexamethasone regulation of marrow stromal-derived osteoblast cells [J]. J Cell Biochem,1996,62:476.
14、Alborzi A, Mac K, Glackin CA,et al. Endochondral and intramembranous fetal bone development:osteoblastic cell proliferation and expression of alkaline phosphatase, mtwist, and histone H4 [J]. J Craniofac Gent Dev Biol,1996,6(2):94.
15、Al GP, SHU YP, YANG GH,et al. Osteogenic and chondrogenic differentiation of mesenchymal cells from swine bone marrow [J]. Zhongguo Linchuang Kangfu(Chin J Clin Rehabil),2002,6(18):2689.
16、JIANG Y, JAHAGIRDAR B N, REINHARDT R L, et al. Pluripotency of mesenchymal stem cells derived from adult marrow [J]. Nature,2002,418:41-49
17、王洪复.骨细胞图谱与骨细胞体外培养技术[M].上海：上海科学技术出版社,2001：60-64.
18、ZHANG B, WANG F, DENG L, et al. Isolating and culturing rat Marrow mesenchymal stem cells and studying their phenotypical and functional properties [J]. Journal of West China University of Medical Sciences,2003,34 (4):738
19、LISIGNOSI G, REMIDDI G, CATTINI L, et al. Anelevatednumber of differentiated osteoblast colonies can be obtained from rat bone marrow stromal cells using a gradient isolation procedure [J]. Connect Tissue Res,2001,42 (1):49.
20、FRIEDENSTEIN A J, CHAILAKHYAN R K, GERASIMOV U V. Bone marrow osteogenic stem cells:in vitro cultivation and transplantation in diffusion chambers [J] Cell Tissue Kinet,1987,20 (3):26-27.
21、刘斌钰,李宁毅,樊功为,等.大鼠骨髓间充质干细胞的培养鉴定及向成骨细胞诱导分化研究[J].齐鲁医学杂志,2007,22(3)：215-217.
22、DERYUGINA E L, MULLER-SIEBURG C E. Stromal cells in long term cultures: keys to the elucidation of hematopoietic development [J]. Crit Rev Immunol, 1993,13:115-120.
23、PEISTER A, MELLAD J A, LARSON B L. Adult stem cells from bone marrow (MSCS) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential [J]. Blood,2004,103 (5):1662-1668.
24、GUO Z, YANG J, LIU X, et al. Biological features of mesenchymal stem cells from human bone marrow [J].Chin Med J (Engl),2001,114 (9):950-953
25、MANIATOPOULOS C, SODEK J, MELCHER A H. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats [J]. Cell Tissue Res, 1988,254 (2):314-317.
26、陈光兴,杨柳,周红星,等.间充质细胞与骨组织共培养条件下的成骨特性[J].中国临床康复,2004,8(8)：1433-1435
27、阮绪芝,陈霞萍,严世荣,等.骨髓基质干细胞的分离纯化及培养[J].生物学杂志,2004,21(2)：25-27.
28、KNIPPENBERG M, HELDER M N, DOULABI B Z, et al. Adipose tissue-derived mesenchymal stem cells acquire bone cell-like responsiveness to fluid shear stress on osteogenic stim-ulation[J]. Tissue Engin,2005,11 (11-12):1780-1788.
29、LIN H T, TARNG Y W, CHEN Y C, et al. Using human plasma supplemented medium to cultivate human bone mar-row-derived mesenchymal stem cell and evaluation of its multi-ple-lineage potential [J]. Transplant Proc,2005,37 (10):4504-4505.
30、 张文,田杰,江德勤,等.骨髓MSC体外分化为心肌样细胞相关调控基因的时序表达[J].中华心血管病杂志,2004,32(11)：1004-1008.
31、KITANO Y,'RADU A, SHABBAN A, et al. Selection, en-richment and culture expansion of murine mesenchymal pro-genitor cells by retroviral transduction of cycline adherent bone marrow cells [J]. Exp Hematol,2000,28 (12):1460-1469
32、GUO X M, WANG C Y, WANG Y H, et al. Experimental study of the isolation, culture and in chondrogenic differentia-tion of human bone mesenchymal stem cell [J] Chin J Stoma-tol,2003,38 (1):63-66.
33、WULF G G, JACKSON K A, GOODELL M A. Somatic stem cell plasticity: current evidence and emerging concepts [J]. Exp Hematol,2001,29 (12):1361-1370.
34、BARRY F P. Mesenchynal stem cell therapy in joint disease [J]. Novartis Found Symp,2003,249:86-96.
35、Pittenger MF, Packay AM, Beck SC, et al Mutilinage potential of adult human mesenchymal stem cell (J) Science,1999; 284(5411):143 7
36、Rockop DJ Marrow stromal cells as stem cells form nonhematopoietic tissues (J) Science,1997; 276(5309)71 4
37、Kopen GC, Prockop DJ, Phinney DG Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains (J) Proc Natl Acad Sci USA,1999; 96(19):10711 6
38、Brazelton TR, Rossi FMV, Keshet GI, et al From marrow to brain:expression of neuronal phenotypes in adult mice (J) Science,2000; 290(5497):1775 9
39、Ankeny DP,McTigue DM, Jakeman LB,et al Bone marrow transplants provide tissue protection and directional guidance for axons after contusive spinal cord injury in rats (J) Exp Neurol,2004; 190(1):17 31
40、Hofstetter CP, Schwarz EJ, Hess D, et al Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery (J) PNAS,2002; 99(4):2199 204
41、丁亮华,罗光华,董选.成人骨髓基质干细胞体外诱导成骨细胞＼[J＼].江苏医药,2005,2：84-86.